Cyclic ether vitamin D3 compounds, 1α(OH) 3-epi-vitamin D3 compounds and uses thereof

ABSTRACT

Novel cyclic ether vitamin D3 compounds having a cyclic ether side chain are disclosed. These compounds were first identified as metabolites of 3-epi vitamin D3 produced via a tissue-specific metabolic pathway which catalyzes the formation of a cyclic ether structure. Also disclosed are 1α(OH) 3-epi vitamin D3 compounds, which are produced via the epimerization of a 3-β-hydroxyl group of 1α(OH) vitamin D3 precursor in vivo. The vitamin D3 compounds of the present invention can be used as substitutes for natural and synthetic vitamin D3 compounds.

RELATED APPLICATIONS

This application claims priority to U.S. provisional applicationApplication No. 60/046,690 filed on May 16, 1997, the contents of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

The importance of the vitamin D in the biological systems of higheranimals has been recognized since its discovery by Mellanby in 1920(Mellanby, E. (1921) Spec. Rep. Ser. Med. Res. Council (GB) SRS 61:4).It was in the interval of 1920-1930 that vitamin D officially becameclassified as a "vitamin" that was essential for the normal developmentof the skeleton and maintenance of calcium and phosphorous homeostasis.

Studies involving the metabolism of vitamin D₃ (cholecalciferol) wereinitiated with the discovery and chemical characterization of the plasmametabolite, 25-hydroxyvitamin D₃ [25(OH)D₃ ] (Blunt, J. W. et al. (1968)Biochemistry 6:3317-3322) and the hormonally active form, 1α,25(OH)₂ D₃(Myrtle, J. F. et al. (1970) J. Biol. Chem. 245:1190-1196; Norman, A. W.et al. (1971) Science 173:51-54; Lawson, D. E. M. et al (1971) Nature230:228-230; Holick, M. F. (1971) Proc. Natl. Acad. Sci. USA68:803-804). The formulation of the concept of a vitamin D endocrinesystem was dependent both upon appreciation of the key role of thekidney in producing 1α, 25(OH)₂ D₃ in a carefully regulated fashion(Fraser, D. R. and Kodicek, E (1970) Nature 288:764-766; Wong, R. G. etal. (1972) J. Clin. Invest. 51:1287-1291), and the discovery of anuclear receptor for 1α,25(OH)₂ D₃ (VD₃ R) in the intestine (Haussler,M. R. et al. (1969) Exp. Cell Res. 58:234-242; Tsai, H. C. and Norman,A. W. (1972) J. Biol. Chem. 248:5967-5975). The operation of the vitaminD endocrine system depends on the following: first, on the presence ofcytochrome P450 enzymes in the liver (Bergman, T. and Postlind, H.(1991) Biochem. J. 276:427-432; Ohyama, Y and Okuda, K. (1991) J. Biol.Chem. 266:8690-8695) and kidney (Henry, H. L. and Norman, A. W. (1974)J. Biol. Chem. 249:7529-7535; Gray, R. W. and Ghazarian, J. G. (1989)Biochem. J. 259:561-568), and in a variety of other tissues to effectthe conversion of vitamin D₃ into biologically active metabolites suchas 1α,25(OH)₂ D₃ and 24R,25(OH)₂ D₃ ; second, on the existence of theplasma vitamin D binding protein (DBP) to effect the selective transportand delivery of these hydrophobic molecules to the various tissuecomponents of the vitamin D endocrine system (Van Baelen, H. et al.(1988) Ann NY Acad. Sci. 538:60-68; Cooke, N. E. and Haddad, J. G.(1989) Endocr. Rev. 10:294-307; Bikle, D. D. et al. (1986) J. Clin.Endocrinol. Metab. 63:954-959); and third, upon the existence ofstereoselective receptors in a wide variety of target tissues thatinteract with the agonist 1α,25(OH)₂ D₃ to generate the requisitespecific biological responses for this secosteroid hormone (Pike, J. W.(1991) Annu. Rev. Nutr. 11:189-216). To date, there is evidence thatnuclear receptors for 1α,25(OH)₂ D₃ (VD₃ R) exist in more than 30tissues and cancer cell lines (Reichel, H. and Norman, A. W. (1989)Annu. Rev. Med. 40:71-78).

Vitamin D₃ and its hormonally active forms are well-known regulators ofcalcium and phosphorous homeostasis. These compounds are known tostimulate, at least one of, intestinal absorption of calcium andphosphate, mobilization of bone mineral, and retention of calcium in thekidneys. Furthermore, the discovery of the presence of specific vitaminD receptors in more than 30 tissues has led to the identification ofvitamin D₃ as a pluripotent regulator outside its classical role incalcium/bone homeostasis. A paracrine role for 1α,25(OH)₂ D₃ has beensuggested by the combined presence of enzymes capable of oxidizingvitamin D₃ into its active forms, e.g., 25-OHD-1α-hydroxylase, andspecific receptors in several tissues such as bone, keratinocytes,placenta, and immune cells. Moreover, vitamin D₃ hormone and activemetabolites have been found to be capable of regulating cellproliferation and differentiation of both normal and malignant cells(Reichel, H. et al. (1989) Ann. Rev. Med. 40: 71-78).

Given the pluripotent activities of vitamin D₃ and its metabolites, muchattention has focused on the development of synthetic analogs of thesecompounds. However, clinical applications of vitamin D₃ and itsstructural analogs have been limited by the undesired side effectselicited by these compounds after administration to a subject, such asthe deregulation of calcium and phosphorous homeostasis in vivo thatresults in hypercalcemia.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofvitamin D3 compounds having a cyclic ether side chain, referred tohereinafter as "cyclic ether vitamin D3 compounds", and which arerepresented by the formula I. This invention also describes 3-epi formsof 1α-hydroxy-vitamin D3 compounds, which are represented by the formulaII. The cyclic ether and 1α-hydroxy-vitamin D3 compounds of formulas Iand II, respectively, referred to hereinafter as "vitamin D3 compoundsof formulas I and II" can be produced in vivo via a pathway whichcatalyzes the epimerization 3-β-hydroxy-vitamin D3 in certain tissues,e.g., keratinocytes, bone cells. The vitamin D3 compounds of the presentinvention can be used as substitutes for natural and synthetic forms ofvitamin D3.

Accordingly, the present invention pertains to cyclic ether vitamin D3compounds having the formula (I) as follows: ##STR1## wherein A₁, A₂ andA₃ represent a single or a double bond; X, R₁, R₂, R₃, R₄ and R₅ can,e.g., be chosen individually from the group of: a hydrogen, a halogen, ahaloalkyl, a hydroxy, a hydroxy-protecting group, an alkyl, e.g., alower alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl group and aheterocyclic group. The orientation of the X group can be in either anα- or a β-configuration.

In a preferred embodiment, the cyclic ether vitamin D3 compound is inits 3-epi configuration, wherein the orientation of the X group on theA-ring is in an α-configuration.

The present invention also pertains to 3-epi forms of 1α-hydroxy-vitaminD3 compounds having the formula II as follows: ##STR2## wherein A₁represents a single, a double, e.g., a trans-double, a cis-double, or atriple bond; A₂, A₃ and A₄ represent a single or a double bond; R₂, R₃,R₄, R₇, R₈ and R₉ can, e.g., be chosen individually from the group of: ahydrogen, a deuterium, a deuteroalkyl, a hydroxy, an alkyl, e.g., alower alkyl, e.g., a C₁ -C₄ alkyl, an alkoxide, an O-acyl, a halogen,e.g., a fluoride, a haloalkyl (e.g., a fluoroalkyl, --CF₃), ahydroxyalkyl, e.g., a hydroxyalkyl wherein the alkyl group is a C₄ -C₁₀alkyl, an amine or a thiol group, and wherein the pairs of R₂ and R₃, orR₄ and R₇ taken together can be an oxygen atom, e.g., as in a carbonylmoiety ##STR3## and R₅ and R₆ can, e.g., each be chosen individuallyfrom the group of: a hydrogen, a deuterium, a halogen, e.g., a fluoride,an alkyl, e.g., a lower alkyl, e.g., a C₁ -C₄ alkyl, a hydroxyalkyl, ahaloalkyl, e.g., a fluoroalkyl, and a deuteroalkyl. The amine or thiolgroup of R₂, R₃, R₄, R₇, R₈ and R₉ can be substituted to form, e.g., aprimary or a secondary amine, or a primary or secondary thiol, whereinthe substituents can be an alkyl or an aryl group, e.g., a substituenthaving 2- to 10-carbon atoms.

In another aspect, the present invention further pertains to apharmaceutical composition comprising, a therapeutically effectiveamount of a vitamin D3 compound having the formulas I or II, and apharmaceutically acceptable carrier.

In yet another aspect, this invention provides a method of modulating abiological activity of a vitamin D3-responsive cell. This methodcomprising contacting the cell with an effective amount of an isolatedvitamin D3 compound of formulas I and II such that modulation of theactivity of the cell occurs.

Another aspect of the invention provides a method of treating in asubject, a disorder characterized by aberrant growth or activity of acell, comprising administering to the subject an effective amount of apharmaceutical composition of a vitamin D3 compound of formulas I and IIsuch that the growth or activity of the cell is reduced.

In a preferred embodiment, the vitamin D3 compound of formulas I and IIused in the treatment has improved biological properties compared tovitamin D3, such as enhanced stability and/or reduced toxicity.

In one aspect, a method for inhibiting the proliferation and/or aninducing the differentiation of a hyperproliferative skin cell isprovided, wherein the hyperproliferative skin cell can be an epidermalcell or an epithelial cell. Accordingly, therapeutic methods fortreating hyperproliferative skin disorders, e.g., psoriasis, areprovided.

In certain embodiments, the instant method can be used for the treatmentof, or prophylactic prevention of a disorder characterized by aberrantcell growth of vitamin D3-responsive neoplastic cell, e.g., byadministering a pharmaceutical preparation of a vitamin D3 compoundhaving the formula as shown in I or II in an amount effective to inhibitgrowth of the neoplastic cells.

In another aspect, the subject method can be used to modulate an immuneresponse, comprising administering to a subject a pharmaceuticalpreparation of a vitamin D compound so as to alter immune function inthe subject. In one embodiment, the method can be used in the treatmentof lymphoid cells, e.g., T cells, natural killer cells, so as tosuppress immune reactions, e.g., to decrease T cell activity, e.g., todecrease production of lymphokines such as IL-2 and IFN-γ, to decrease Tcell proliferation. In preferred embodients, the method can be used intreating graft rejection, autoimmunity and inflammation.

In yet another aspect, the vitamin D3 compound of the present inventionare useful in the treatment of disorder characterized by a deregulationof calcium and phosphate metabolism, comprising administering to asubject a pharmaceutical preparation of a vitamin D3 compounds offormulas I and II so as to ameliorate the deregulation in calcium andphosphate metabolism.

In a preferred embodiment, the disorder is osteoporosis. In otherembodiments, the vitamin D3 compounds of formulas I and II can be usedto treat diseases characterized by other deregulations in the metabolismof calcium and phosphate.

In another aspect, a method for inhibiting PTH secretion in parathyroidcell using the vitamin D3 compound of formulas I and II is provided.Furthermore, therapeutic methods for treating secondaryhyperparathyroidism are also provided.

In yet another aspect, the present invention provides a method ofpreventing or protecting against neuronal loss by contacting a vitaminD3-responsive cell, e.g., a neuronal cell, with a vitamin D3 compound offormulas I and II to prevent or retard neuron loss.

In yet another aspect, the present invention provides a method ofmodulating the activity of a vascular smooth muscle cell by contacting avitamin D3-responsive smooth muscle cell with a vitamin D3 compound offormulas I and II to activate or, preferably, inhibit the activity ofthe cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a compilation of the chemical structures of 266 vitamin D₃compounds (Boullion, R. et al. (1995) Endocrinology Reviews 16(2):200-257, the contents of which including the figures depicted thereinare incorporated by reference). Each analog is identified by itschemical name and a one, two, or three-letter identification code.

FIG. 2 shows the HPLC profile and UV spectra of the metabolites producedin human keratinocytes incubated with 1α, 25(OH)₂ -3-epi vitamin D₃.

FIG. 3 shows the mass spectra of 1α, 25(OH)₂ -3-epi vitamin D₃ and itscyclic ether metabolite.

FIG. 4 shows the proposed metabolic pathway for the formation of thecyclic ether metabolite of 1α, 25(OH)₂ -3-epi vitamin D₃.

FIG. 5A shows the metabolism of 1α(OH)-vitamin D₃ into its 3 epi form inthe rat osteosarcoma cell line (UMR-106).

FIG. 5B is a schematic of the 3-epimerization of 1α(OH)D₃ into1α(OH)-3-epi vitamin D₃.

FIG. 6 shows the mass spectra of 1α(OH)D₃ and its 3-epi metabolite.

FIG. 7 shows the HPLC profile and UV spectra of the metabolites producedin rat osteosarcoma cell lines (UMR-106) which were incubated with1α(OH)D₃ for 24, 48, or 84 hours.

DETAILED DESCRIPTION OF THE INVENTION

The language "cyclic ether vitamin D3 compound" is intended to includeall vitamin D3 compounds having a cyclic ether side chain, including3-epimeric and non-3-epimeric of vitamin D3 as represented by thegeneral formula I.

As used herein, the terms "3-epi vitamin D3" or "3-epi D3" compounds areintended to include vitamin D3 compounds having a substituent, e.g., afunctional group, e.g., a hydroxyl group, attached to the carbon atposition 3 of the A-ring in an α-configuration rather than aβ-configuration. The language "3-epi forms of 1α-hydroxy-vitamin D3compounds" or "1α-hydroxy-3-epi-vitamin D3 compounds" is intended toinclude 1α-hydroxy-vitamin D3 compounds having the hydroxyl group,attached to the carbon at position 3 of the A-ring in an α-configurationrather than a β-configuration, and which are represented by the generalformula II as described in detail below.

The cyclic ether and 1α-hydroxy-vitamin D3 compounds of formulae I andII, respectively, referred to hereinafter as "vitamin D3 compounds offormulas I and II" can be produced in vivo via a pathway which catalyzesthe epimerization 3-β-hydroxy-vitamin D3 in certain tissues, e.g.,keratinocytes or bone cells.

The language "vitamin D3 compounds" or "cholecalciferols" (also referredto herein as "D3 compounds") is intended to include compounds which arestructurally similar to vitamin D₃. Many of these compounds areart-recognized and comprise a large number of natural precursors,metabolites, as well as synthetic analogs of the hormonally active1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂ D₃). This language is intended toinclude vitamin D₃, or an analog thereof, at any stage of itsmetabolism, as well as mixtures of different metabolic forms of vitaminD₃ or analogs thereof. Furthermore, the term "vitamin D₃ compound" alsoincludes synthetic analogs of vitamin D₃ illustrated in FIG. 1.

In the formulas presented herein, the various substituents areillustrated as joined to the steroid nucleus by one of these notations:a dotted line (---) indicating a substituent which is in theβ-orientation (i.e., above the plane of the ring), a wedged solid line() indicating a substituent which is in the α-orientation (i.e., belowthe plane of the molecule), or a solid line (--) indicating asubstituent in the plane of the ring. It should be understood that thestereochemical convention in the steroid field is opposite from thegeneral chemical field, wherein a dotted line indicates a substituentwhich is in an α-orientation (i.e., below the plane of the molecule),and a wedged solid line indicates a substituent which is in theβ-orientation (i.e., above the plane of the ring). As shown, the A ringof the hormone 1α,25(OH)₂ D₃ contains two asymetric centers at chiralcarbons-1 and -3, each one containing a hydroxyl group inwell-characterized configurations, namely the 1α- and 3β-hydroxylgroups.

Accordingly, the present invention pertains to cyclic ether vitamin D3compounds having the formula (I) as follows: ##STR4## wherein A₁, A₂ andA₃ represent a single or a double bond; X, R₁, R₂, R₃, R₄ and R₅ can,e.g., be chosen individually from the group of: a hydrogen, a halogen, ahaloalkyl, a hydroxy, a hydroxy-protecting group, an alkyl, e.g., alower alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl group and aheterocyclic group. The orientation of the X group can be in either anα- or a β-configuration.

In a preferred embodiment, the cyclic ether vitamin D3 compound isrepresented by the general formula I, wherein the orientation of the Xgroup on the A-ring is in an α-configuration; A₁ is a single bond; A₂and A₃ are each a double bond; --X and R₁ are hydroxyl groups; R₂, R₃,R₄ and R₅ are a hydrogen.

The present invention also pertains to 3-epi forms of 1α-hydroxy-vitaminD3 compounds having the formula II: ##STR5## wherein A₁ represents asingle, a double, e.g., a trans-double, a cis-double, or a triple bond;A₂, A₃ and A4 represent a single or a double bond; R₂, R₃, R₄, R₇, R₈and R₉ can, e.g., be chosen individually from the group of: a hydrogen,a deuterium, a deuteroalkyl, a hydroxy, an alkyl, e.g., a lower alkyl,e.g., a C₁ -C₄ alkyl, an alkoxide, an O-acyl, a halogen, e.g., afluoride, a haloalkyl (e.g., a fluoroalkyl, --CF₃), a hydroxyalkyl,e.g., a hydroxyalkyl wherein the alkyl group is a C₄ -C₁₀ alkyl, anamine or a thiol group, and wherein the pairs of R₂ and R₃, or R₄ and R₇taken together can be an oxygen atom, e.g., as in a carbonyl moiety##STR6## and R₅ and R₆ can, e.g., each be chosen individually from thegroup of: a hydrogen, a deuterium, a halogen, e.g., a fluoride, analkyl, e.g., a lower alkyl, e.g., a C₁ -C₄ alkyl, a hydroxyalkyl, ahaloalkyl, e.g., a fluoroalkyl, and a deuteroalkyl. The amine or thiolgroup of R₂, R₃, R₄, R₇, R₈ and R₉ can be substituted to form, e.g., aprimary or a secondary amine, or a primary or a secondary thiol, whereinthe substituents can be an alkyl or an aryl group, e.g., a substituenthaving 2- to 10-carbon atoms.

In a preferred embodiment, A₁, A₂ and A₃ are each a single bond; A₄ is adouble bond; R₂, R₃, R₅, R₆, R₈ and R₉ are each a hydrogen or an alkyl,e.g., a methyl; and R₄ and R₇ are each a hydrogen, a hydroxy or analkyl, e.g., a lower alkyl, e.g., a methyl or an ethyl group. Thechirality of the positions substituted by R₄ and R₇ can be in either anR- or an S-configuration.

Exemplary preferred 1α-hydroxy vitamin D₃ compounds encompassed byformula II include: 1α hydroxy 3-epi vitamin D₃, 1α,24 dihydroxy 3-epivitamin D₃ (both 1α, 24R-dihydroxy 3-epi vitamin D₃ and 1α,24S-dihydroxy 3-epi vitamin D₃), 1α hydroxy 24-ethyl 3-epi vitamin D₃,1α hydroxy 24-methyl 3-epi vitamin D₃ and 1α, 24-dihydroxy 24-methyl3-epi vitamin D₃ having the following chemical formulae: ##STR7##

A representation of 1α-hydroxy-vitamin D3 prior to 3-epi conversion isalso depicted as analog BP in FIG. 1.

In yet another embodiment, the present invention provides isolatedvitamin D3 compounds of formulae I and II, having at least onebiological activity of vitamin D3, and having improved biologicalproperties compared to vitamin D3, such as enhanced stability in vivoand/or reduced toxicity.

The term "epimer" or "epi" compounds is intended to include compoundshaving a chiral carbon that varies in the orientation of a single bondto a substituent on that carbon compared to the naturally-occurring (orreference) compound, for example, a carbon where the orientation of thebond to the substituent is in an α-configuration, instead of aβ-configuration. The 3-epimer forms of vitamin D3 compounds having thegeneral formulas I and II have a substituent, e.g., a hydroxyl group,attached to the carbon at position 3 of the A-ring in an α-configurationrather than a β-configuration, whereas all other substituents can be ineither an α- or a β-configuration.

As used herein, the term "substituent" refers to a moiety, for example afunctional group, attached to the carbon position 3 of the A ring of thevitamin D₃ compound that allows the compound to perform its intendedfunction. Accordingly, the term "substituent" is intended to includehydrogen, halogen, haloalkyl, hydroxy, hydroxy-protecting group, alkyl,e.g. lower alkyl, alkenyl, e.g., lower alkenyl, alkynyl, e.g., loweralkynyl, alkoxy, aryl group and heterocyclic group.

The term "chiral" refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term"achiral" refers to molecules which are superimposable on their mirrorimage partner. The term "stereoisomers" or "isomers" refer to compoundswhich have identical chemical constitution, but differ with regard tothe arrangement of the atoms or groups in space. In particular,"enantiomers" refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another. An equimolar mixture oftwo enantiomers is called a "racemic mixture" or a "racemate"."Diastereomers" refer to stereoisomers with two or more centers ofdissymmetry and whose molecules are not mirror images of one another.With respect to the nomenclature of a chiral center, terms "d" and "l"configuration are as defined by the IUPAC Recommendations. As to the useof the terms, diastereomer, racemate, epimer and enantiomer will be usedin their normal context to describe the stereochemistry of preparations.

As used herein, the language "isomeric counterparts of vitamin D3" or"non-epimeric forms" refers to stereoisomers of the 3-epi vitamin D3compounds. For example, vitamin D3 compounds which have the orientationof the 3-hydroxy group in a β-configuration.

The terms "isolated" or "substantially purified" as used interchangeablyherein refer to vitamin D₃ compounds in a non-naturally occurring state.The compounds can be substantially free of cellular material or culturemedium when naturally produced, or chemical precursors or otherchemicals when chemically synthesized. In other preferred embodiments,the terms "isolated" or "substantially purified" also refer topreparations of a chiral compound which substantially lack one of theenantiomers, i.e., enantiomerically enriched or non-racemic preparationsof a molecule. Similarly, isolated epimers or diasteromers refers topreparations of chiral compounds which are substantially free of otherstereochemical forms. For instance, isolated or substantially purifiedvitamin D₃ compounds includes synthetic or natural preparations of avitamin D₃ enriched for the stereoisomers having a substituent attachedto the chiral carbon at position 3 of the A-ring in an α-configuration,and thus substantially lacking other isomers having a β-configuration.Unless otherwise specified, such terms refer to vitamin D₃ compositionsin which the ratio of α to β forms is greater that 1:1 by weight. Forinstance, an isolated preparation of an α epimer means a preparationhaving greater than 50% by weight of the α-epimer relative to the βstereoisomer, more preferably at least 75% by weight, and even morepreferably at least 85% by weight. Of course the enrichment can be muchgreater than 85%, providing a "substantially epimer enriched", whichrefers to preparations of a compound which have greater than 90% of theα-epimer relative to the β stereoisomer, and even more preferablygreater than 95%. The term "substantially free of the β stereoisomer"will be understood to have similar purity ranges.

As used herein, the language "alkyl" is art-recognized and includes tothe radical of saturated aliphatic groups, including straight-chainalkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic)groups, alkyl substituted cycloalkyl groups, and cycloalkyl substitutedalkyl groups. In preferred embodiments, a straight chain or branchedchain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C₁ -C₃₀for straight chain, C₃ -C₃₀ for branched chain), and more preferably 20or fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms intheir ring structure, and more preferably have 5, 6 or 7 carbons in thering structure.

Unless the number of carbons is otherwise specified, "lower alkyl" asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six, and most preferablyfrom one to four carbon atoms in its backbone structure, which may bestraight or branched-chain, which may be straight or branched-chain.Examples of lower alkyl groups include methyl, ethyl, n-propyl,i-propyl, tert.-butyl, hexyl, heptyl, octyl and so forth. Likewise,"lower alkenyl" and "lower alkynyl" have similar chain lengths.Preferred alkyl groups include lower alkyls. Examples of alkylene groupsare methylene, ethylene, propylene and so forth.

Moreover, the term alkyl as herein is intended to include both"unsubstituted alkyls" and "substituted alkyls", the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, halogen, hydroxyl, carbonyl (including aldehydes,ketones, carboxylates, and esters), alkoxyl, ether, phosphoryl, cyano,amino, acylamino, amido, amidino, imino, sulfhydryl, alkylthio,arylthio, thiolcarbonyl (including thiolformates, thiolcarboxylic acids,and thiolesters), sulfonyl, nitro, heterocyclyl, aralkyl, or an aromaticor heteroaromatic moiety. It will be understood by those skilled in theart that the moieties substituted on the hydrocarbon chain canthemselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, acylaminos, iminos, amidos, phosphoryls(including phosphonates and phosphinates), sulfonyls (includingsulfates, sulfonatos, sulfamoyls, and sulfonamidos), and silyl groups,as well as ethers, alkylthios, arylthios, carbonyls (including ketones,aldehydes, carboxylates, and esters), --CF₃, --CN and the like.Exemplary substituted alkyls are described below. Cycloalkyls can befurther substituted with alkyls, alkenyls, alkoxys, alkylthios,arylthios, aminoalkyls, carbonyl-substituted alkyls, --CF₃, cyano(--CN), and the like.

The term "aralkyl", as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms "alkenyl" and "alkynyl" are art-recognized and include tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

The terms "alkoxyl" is art-recognized and includes to an grouprepresented by the formula --O-alkyl. Representative alkoxyl groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like. Unlessotherwise specified, an "alkoxy" group can be replaced with a grouprepresented by --O-alkenyl, --O-alkynyl, --O-aryl (i.e., an aryloxygroup), or --O-heterocyclyl. An "ether" is two substituted orunsubstituted hydrocarbons covalently linked by an oxygen. Accordingly,the substituent of, e.g., an alkyl that renders that alkyl an ether isor resembles an alkoxyl, such as can be represented by one of --O-alkyl,--O-alkenyl, --O-alkynyl, --O-aryl, or --O-heterocyclyl. The term "loweralkoxy" includes a lower alkyl group attached to the remainder of themolecule by oxygen.

Examples of alkoxy groups include methoxy, ethoxy, isopropoxy,tert.-butoxy and so forth. The term "phenyl alkoxy" refer to an alkoxygroup which is substituted by a phenyl ring. Examples of phenyl alkoxygroups are benzyloxy, 2-phenylethoxy, 4-phenylbutoxy and so forth. Theterm "alkanoyloxy group" refers to the residue of an alkylcarboxylicacid formed by removal of the hydrogen from the hydroxyl portion of thecarboxyl group. Examples of alkanoyloxy groups include formyloxy,acetoxy, butyryloxy, hexanolyoxy and so forth. The term "substituted" asapplied to "phenyl" refers to phenyl which is substituted with one ormore of the following groups: alkyl, halogen (i.e., fluorine, chlorine,bromine or iodine), nitro, cyano, trifluoromethly and so forth. The"alkanol" or a "hydroxyalkyl" refer to a compound derived by protonationof the oxygen atom of an alkoxy group. Examples of alkanols includemethanol, ethanol, 2-propanol, 2-methyl-2-propanol and the like.

As used herein the term "hydroxy-protecting group" includes any groupcommonly used for the protection of hydroxy functions during subsequentreactions, including, for example, acyl or alkylsilyl groups such astrimethylsilyl, triethylsilyl, t-butyldimethylsilyl and analogousalkylated silyl radicals, or alkoxyalkyl groups such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, tetrahydrofuranyl ortetrahydropyranyl. A "protected-hydroxy" is a hydroxy functionderivatized by one of the above hydroxy-protecting groupings.

As used herein, the term "halogen" designates --F, --Cl, --Br or --I;the term "sulfhydryl" or "thiol" means --SH; the term "hydroxyl" means--OH.

The term "aryl" is art-recognized and includes 5- and 6-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Aryl groups also include polycyclic fusedaromatic groups such as naphthyl, quinolyl, indolyl, and the like. Thosearyl groups having heteroatoms in the ring structure may also bereferred to as "aryl heterocycles", "heteroaryls" or "heteroaromatics".The aromatic ring can be substituted at one or more ring positions withsuch substituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, acylamino,azido, nitro, sulfhydryl, imino, amido, amidino, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, arylthio,sulfonyl, sulfonamido, sulfamoyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, --CF₃, --CN, or thelike. Aryl groups can also be fused or bridged with alicyclic orheterocyclic rings which are not aromatic so as to form a polycycle(e.g., tetralin).

The terms "heterocyclyl" or "heterocyclic group" are art-recognized andinclude 3- to 10-membered ring structures, more preferably 4- to7-membered rings, which ring structures include one to four heteroatoms.Heterocyclyl groups include pyrrolidine, oxolane, thiolane, imidazole,oxazole, piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, lactones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,acylamino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, arylthio, sulfonyl, ketone,aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety,--CF₃, --CN, or the like.

The terms "polycyclyl" or "polycyclic group" are art-recognized andinclude two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are "fused rings".Rings that are joined through non-adjacent atoms are termed "bridged"rings. Each of the rings of the polycycle can be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, acylamino,nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, arylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, --CF₃,--CN, or the like.

Vitamin D Synthesis

The vitamin D3 compounds of the present invention can be prepared usinga variety of synthetic methods, as are known in the art. For example,many of the above-described compounds can be prepared by chemicalsynthesis, or alternatively by enzymatic conversion of a 3β-vitamin D3precursor, e.g., by perfusing a 3β-vitamin D3 precursor, a vitamin D3compound having the orientation of the hydroxy group at position 3 ofthe A-ring in a β-configuration, in a tissue-containing an enzyme whichcatalyzes the epimerization of the 3-β-hydroxyl group to the 3α formvitamin D3 compounds, e.g., keratinocytes or bone cells as described inExamples I, II and IV.

For example, methods for synthesizing vitamin D3 compounds of formulas Iand II are well known in the art (see e.g., Bouillon, R. et al.,Endocrine Reviews 16(2):201-204; Ikekawa N. (1987) Med. Res. Rev.7:333-366; DeLuca H. F. and Ostrem V. K. (1988) Prog. Clin. Biol. Res.259:41-55; Ikekawa N. and Ishizuka S. (1992) CRC Press 8:293-316;Calverley M. J. and Jones G. (1992) Academic Press 193-270; Pardo R. andSantelli M. (1985) Bull. Soc. Chim. Fr:98-114; Bythgoe B. (1980) Chem.Soc. Rev. 449-475; Quinkert G. (1985) Synform 3:41-122; Quinkert G.(1986) Synform 4:131-256; Quinkert G. (1987) Synform 5:1-85; Mathieu C.et al. (1994) Diabetologia 37:552-558; Dai H. and Posner G. H. (1994)Synthesis 1383-1398). Exemplary methods of synthesis include thephotochemical ring opening of a 1-hydroxylated side chain-modifiedderivative of 7-dehydrocholesterol which initially produces a previtaminthat is easily thermolyzed to vitamin D3 in a well known fashion (BartonD. H. R. et al. (1973) J. Am. Chem. Soc. 95:2748-2749; Barton D. H. R.(1974) JCS Chem. Comm. 203-204); phosphine oxide coupling methoddeveloped by (Lythgoe et al (1978) JCS Perkin Trans. 1:590-595) whichcomprises coupling a phosphine oxide to a Grundmann's ketone derivativeto directly produce a 1α,25(OH)₂ D3 skeleton as described in BaggioliniE. G. et al. (1986) J. Org. Chem. 51:3098-3108; DeSchrijver J. andDeClercq P. J. (1993) Tetrahed Lett 34:4369-4372; Posner G. H. andKinter C. M. (1990) J. Org. Chem. 55:3967-3969; semihydrogenation ofdienynes to a previtamin structure that undergoes rearrangement to thecorresponding vitamin D3 analog as described by Harrison R. G. et al.(1974) JCS Perkin Trans. 1:2654-2657; Castedo L. et al. (1988) TetrahedLett 29:1203-1206; Mascarenas J. S. (1991) Tetrahedron 47:3485-3498;Barrack S. A. et al. (1988) J. Org. Chem. 53:1790-1796) and Okamura W.H. et al. (1989) J. Org. Chem. 54:4072-4083; the vinylallene approachinvolving intermediates that are subsequently arranged using heat or acombination of metal catalyzed isomerization followed by sensitizedphotoisomerization (Okamura W. H. et al. (1989) J. Org. Chem.54:4072-4083; Van Alstyne E. M. et al. (1994) J. Am. Chem. Soc.116:6207-6210); the method described by Trost et al. B. M. et al. J. Am.Chem. Soc. 114:9836-9845; Nagasawa K. et al. (1991) Tetrahed Lett32:4937-4940 involves an acyclic A-ring precursor which isintramolecular cross-coupled to the bromoenyne leading directly to theformation of 1,25(OH)₂ D3 skeleton; a tosylated derivative which isisomerized to the i-steroid that can be modified at carbon-1 and thensubsequently back-isomerized under sovolytic conditions to form1α,25(OH)₂ D2 or analogs thereof (Sheves M. and Mazur Y. (1974) J. Am.Chem. Soc. 97:6249-6250; Paaren H. E. et al. (1980) J. Org. Chem.45:3253-3258; Kabat M. et al. (1991) Tetrahed Lett 32:2343-2346; WilsonS. R. et al. (1991) Tetrahed Lett 32:2339-2342); the direct modificationof vitamin D derivatives to 1-oxygenated 5,6-trans vitamin D asdescribed in (Andrews D. R. et al. (1986) J. Org. Chem. 51:1635-1637);the Diels-Alders cycloadduct method of previtamin D3 can be used tocyclorevert to 1α,25(OH)₂ D2 through the intermediary of a previtaminform via thermal isomerization (Vanmaele L. et al. (1985) Tetrahedron41:141-144); and, a final method entails the direct modification of1α,25(OH)₂ D2 or an analog through use of suitable protecting groupssuch as transition metal derivatives or by other chemicaltransformations (Okarmura W. H. et al. (1992) J. Cell Biochem.49:10-18). Additional methods for synthesizing vitamins D2 compounds aredescribed in, for example, Japanese Patent Disclosures Nos. 62750/73,26858/76, 26859/76, and 71456/77; U.S. Pat. Nos. 3,639,596; 3,715,374;3,847,955 and 3,739,001.

Examples of the compounds of this invention having a saturated sidechain can be prepared according to the general process illustrated anddescribed in U.S. Pat. No. 4,927,815, the description of which isincorporated herein by reference. Examples of the compounds of thisinvention having an unsaturated side chain is can be prepared accordingto the general process illustrated and described in U.S. Pat. No.4,847,012, the description of which is incorporated herein by reference.Examples of the compounds of this invention wherein R groups togetherrepresent a cyclopentano group can be prepared according to the generalprocess illustrated and described in U.S. Pat. No. 4,851,401, thedescription of which incorporated herein by reference.

Another synthetic strategy for the preparation of side-chain-modifiedanalogues of 1α,25-dihydroxyergocalciferol is disclosed in Kutner etal., The Journal of Organic Chemistry, 1988, 53:3450-3457. In addition,the preparation of 24-homo and 26-homo vitamin D analogs are disclosedin U.S. Pat. No. 4,717,721, the description of which is incorporatedherein by reference.

The enantioselective synthesis of chiral molecules is now state of theart. Through combinations of enantioselective synthesis and purificationtechniques, many chiral molecules can be synthesized as anenantiomerically enriched preparation. For example, methods have beenreported for the enantioselective synthesis of A-ring diastereomers of1α,25(OH)₂ D3 as described in Muralidharan et al. (1993) J. OrganicChem. 58(7): 1895-1899 and Norman et al. (1993) J. Biol. Chem. 268(27):20022-30. Other methods for the enantiomeric synthesis of variouscompounds known in the art include, inter alia, epoxides (see, e.g.,Johnson, R. A.; Sharpless, K. B. In Catalytic Asymmetric Synthesis;Ojima, I., Ed.: VCH: New York, 1993; Chapter 4.1. Jacobsen, E. N. Ibid.Chapter 4.2), diols (e.g., by the method of Sharpless, J. Org. Chem.(1992) 57:2768), and alcohols (e.g., by reduction of ketones, E. J.Corey et al., J. Am. Chem. Soc. (1987) 109:5551). Other reactions usefulfor generating optically enriched products include hydrogenation ofolefins (e.g., M. Kitamura et al., J. Org. Chem. (1988) 53:708);Diels-Alder reactions (e.g., K. Narasaka et al., J. Am. Chem. Soc.(1989) 111:5340); aldol reactions and alkylation of enolates (see, e.g.,D. A. Evans et al., J. Am. Chem. Soc. (1981) 103:2127; D. A. Evans etal., J. Am. Chem. Soc. (1982) 104:1737); carbonyl additions (e.g., R.Noyori, Angew. Chem. Int. Ed. Eng. (1991) 30:49); and ring-opening ofmeso-epoxides (e.g., Martinez, L. E.; Leighton J. L., Carsten, D. H.;Jacobsen, E. N. J. Am. Chem. Soc. (1995) 117:5897-5898). The use ofenymes to produce optically enriched products is also well known in theart (e.g., M. P. Scheider, ed. "Enzymes as Catalysts in OrganicSynthesis", D. Reidel, Dordrecht (1986).

Chiral synthesis can result in products of high stereoisomer purity.However, in some cases, the stereoisomer purity of the product is notsufficiently high. The skilled artisan will appreciate that theseparation methods described herein can be used to further enhance thestereoisomer purity of the vitamin D3-epimer obtained by chiralsynthesis.

Separation of isomers can be accomplished in several ways known in theart. An exemplary straight phase and reverse phase HPLC system used toseparate natural or synthetic diastereomers of 1α,25(OH)₂ D3 is detailedin the appended example and illustrated in FIG. 2. Further methods forseparating a racemic mixture of two enantiomers include chromatographyusing a chiral stationary phase (see, e.g., "Chiral LiquidChromatography", W. J. Lough, Ed. Chapman and Hall, New York (1989)).Enantiomers can also be separated by classical resolution techniques.For example, formation of diastereomeric salts and fractionalcrystallization can be used to separate enantiomers. For the separationof enantiomers of carboxylic acids, the diastereomeric salts can beformed by addition of enantiomerically pure chiral bases such asbrucine, quinine, ephedrine, strychnine, and the like. Alternatively,diastereomeric esters can be formed with enantiomerically pure chiralalcohols such as menthol, followed by separation of the diastereomericesters and hydrolysis to yield the free, enantiomerically enrichedcarboxylic acid. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the isolated vitamin D₃ compounds of formulas Iand II, formulated together with one or more pharmaceutically acceptablecarrier(s).

In a preferred embodiment, these pharmaceutical compositions aresuitable for topical or oral administration to a subject. In otherembodiments, as described in detail below, the pharmaceuticalcompositions of the present invention may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, boluses, powders,granules, pastes; (2) parenteral administration, for example, bysubcutaneous, intramuscular or intravenous injection as, for example, asterile solution or suspension; (3) topical application, for example, asa cream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream or foam; or (5) aerosol,for example, as an aqueous aerosol, liposomal preparation or solidparticles containing the compound.

In certain embodiments, the subject is a mammal, e.g., a primate, e.g.,a human. As used herein, the language "subject" is intended to includehuman and non-human animals. Preferred human animals include a humanpatient having a disorder characterized by the aberrant activity of avitamin D₃ -responsive cell. The term "non-human animals" of theinvention includes all vertebrates, e.g., mammals and non-mammals, suchas non-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

The phrase "therapeutically-effective amount" as used herein means thatamount of a vitamin D₃ compound(s) of formulas I and II, or compositioncomprising such a compound which is effective for the compound toproduce its intended function, e.g., the modulation of activity of avitamin D₃ -response cell. The effective amount can vary depending onsuch factors as the type of cell growth being treated or inhibited, theparticular type of vitamin D₃ compound, the size of the subject, or theseverity of the undesirable cell growth or activity. One of ordinaryskill in the art would be able to study the aforementioned factors andmake the determination regarding the effective amount of the vitamin D₃compound of formulas I and II without undue experimentation.

In certain embodiments, one or more vitamin D₃ compounds as representedby formulas I and II may be administered alone, or as part ofcombinatorial therapy. For example, the vitamin D₃ compounds can beconjointly administered with one or more agents such as mitoticinhibitors, alkylating agents, antimetabolites, nucleic acid,intercalating agents, topoisomerase inhibitors, agents which promoteapoptosis, and/or agents which modulate immune responses. The effectiveamount of vitamin D₃ compound used can be modified according to theconcentrations of the other agents used.

In vitro assay using keratinocytes or parathyroid cells, or an assaysimilar thereto (e.g., differing in choice of cells, e.g., bone cells,intestinal cells, neoplastic cells) can be used to determine an"effective amount" of the vitamin D₃ compounds of formulas I and II, orcombinations thereof. The ordinarily skilled artisan would select anappropriate amount of each individual compound in the combination foruse in the aforementioned in vitro assay or similar assays. Changes incell activity or cell proliferation can be used to determine whether theselected amounts are "effective amount" for the particular combinationof compounds. The regimen of administration also can affect whatconstitutes an effective amount. As described in detail below, vitaminD₃ compounds of formulas I and II can be administered to the subjectprior to, simultaneously with, or after the administration of the otheragent(s). Further, several divided dosages, as well as staggereddosages, can be administered daily or sequentially, or the dose can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic situation.

The phrase "pharmaceutically acceptable" is employed herein to refer tothose vitamin D₃ compounds of formulas I and II, compositions containingsuch compounds, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable carrier" as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be "acceptable" in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions containing the vitamin D₃ compounds of the presentinvention include those suitable for oral, nasal, topical (includingbuccal and sublingual), rectal, vaginal, aerosol and/or parenteraladministration. The compositions may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect. Generally, out ofone hundred per cent, this amount will range from about 1 per cent toabout ninety-nine percent of active ingredient, preferably from about 5per cent to about 70 per cent, most preferably from about 10 per cent toabout 30 per cent.

Methods of preparing these compositions include the step of bringinginto association a vitamin D₃ compound(s) of formulas I and II with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing intoassociation a vitamin D₃ compound with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Compositions of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a vitamin D₃ compound(s)of formulas I and II as an active ingredient. A compound may also beadministered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the vitamin D₃compound(s) of the invention include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active vitamin D₃ compound(s) maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Pharmaceutical compositions of the invention for rectal or vaginaladministration may be presented as a suppository, which may be preparedby mixing one or more vitamin D₃ compound(s) of formulas I and II withone or more suitable nonirritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the rectum or vaginal cavityand release the active agent.

Compositions of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a vitaminD₃ compound(s) of formulas I and II include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive vitamin D₃ compound may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition tovitamin D₃ compound(s) of formulas I and II, excipients, such as animaland vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a vitamin D₃ compound(s)of formulas I and II, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

The vitamin D₃ compound(s) of formulas I and II can be alternativelyadministered by aerosol. This is accomplished by preparing an aqueousaerosol, liposomal preparation or solid particles containing thecompound. A nonaqueous (e.g., fluorocarbon propellant) suspension couldbe used. Sonic nebulizers are preferred because they minimize exposingthe agent to shear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a vitamin D₃ compound(s) of formulas I and II to the body.Such dosage forms can be made by dissolving or dispersing the agent inthe proper medium. Absorption enhancers can also be used to increase theflux of the peptidomimetic across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe peptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more vitamin D₃ compound(s) of formulas Iand II in combination with one or more pharmaceutically-acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofvitamin D₃ compound(s) of formulas I and II in biodegradable polymerssuch as polylactide-polyglycolide. Depending on the ratio of drug topolymer, and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the vitamin D₃ compound(s) of the present invention areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

The term "administration," is intended to include routes of introducinga subject the 3-epimer vitamin D₃ compound of formula I to perform theirintended function. Examples of routes of administration which can beused include injection (subcutaneous, intravenous, parenterally,intraperitoneally, intrathecal, etc.), oral, inhalation, rectal andtransdermal. The pharmaceutical preparations are of course given byforms suitable for each administration route. For example, thesepreparations are administered in tablets or capsule form, by injection,inhalation, eye lotion, ointment, suppository, etc. administration byinjection, infusion or inhalation; topical by lotion or ointment; andrectal by suppositories. Oral administration is preferred. The injectioncan be bolus or can be continuous infusion. Depending on the route ofadministration, the vitamin D₃ compound of formulas I and II can becoated with or disposed in a selected material to protect it fromnatural conditions which may detrimentally effect its ability to performits intended function. The vitamin D₃ compound of formulas I and II canbe administered alone, or in conjunction with either another agent asdescribed above or with a pharmaceutically acceptable carrier, or both.The vitamin D₃ compound can be administered prior to the administrationof the other agent, simultaneously with the agent, or after theadministration of the agent. Furthermore, the vitamin D₃ compound offormulas I and II can also be administered in a proform which isconverted into its active metabolite, or more active metabolite in vivo.

The phrases "parenteral administration" and "administered parenterally"as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases "systemic administration," "administered systemically","peripheral administration" and "administered peripherally" as usedherein mean the administration of a vitamin D₃ compound(s) of formulas Iand II, such that it enters the patient's system and, thus, is subjectto metabolism and other like processes, for example, subcutaneousadministration.

These vitamin D₃ compound(s) of formulas I and II may be administered toa "subject", e.g., mammals, e.g., humans and other animals.Administration can be carried out by any suitable route ofadministration, including orally, nasally, as by, for example, a spray,rectally, intravaginally, parenterally, intracisternally and topically,as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the vitamin D₃compound(s) of formulas I and II, which may be used in a suitablehydrated form, and/or the pharmaceutical compositions of the presentinvention, are formulated into pharmaceutically-acceptable dosage formsby conventional methods known to those of skill in the art.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of this invention may bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. Exemplary dose range is from 0.1 to 10 μg per day.

Uses of the Vitamin D Compounds of the Invention

Another aspect of the invention pertains to isolated vitamin D₃compounds of formulas I and II having at least one biological activityof vitamin D₃, and having improved biological properties whenadministered into a subject than vitamin D₃ under the same conditions,as well as, methods of testing and using these compounds to treatdisorders involving an aberrant activity of a vitamin D3-responsivecell, e.g., neoplastic cells, hyperproliferative skin cells, parathyroidcells, immune cells and bone cells, among others.

The language "biological activities" of vitamin D₃ is intended toinclude all activities elicited by vitamin D₃ compounds of formulas Iand II in a responsive cell. This term includes genomic and non-genomicactivities elicited by these compounds (Bouillon, R. et al. (1995)Endocrinology Reviews 16(2):206-207; Norman A. W. et al. (1992) J.Steroid Biochem Mol. Biol 41:231-240; Baran D. T. et al. (1991) J. BoneMiner Res. 6:1269-1275; Caffrey J. M. and Farach-Carson M. C. (1989) J.Biol. Chem. 264:20265-20274; Nemere I. et al. (1984) Endocrinology115:1476-1483).

As used herein, the term "vitamin D₃ -responsive cell" includes any cellwhich is is capable of responding to a vitamin D₃ compound having theformula I or II, and is associated with disorders involving an aberrantactivity of hyperproliferative skin cells, parathyroid cells, neoplasticcells, immune cells, and bone cells. These cells can respond to vitaminD₃ activation by triggering genomic and/or non-genomic responses thatultimately result in the modulation of cell proliferation,differentiation survival, and/or other cellular activities such ashormone secretion. In a preferred embodiment, the ultimate responses ofa cell are inhibition of cell proliferation and/or induction ofdifferentiation-specific genes. Exemplary vitamin D₃ responsive cellsinclude immune cells, bone cells, neuronal cells, endocrine cells,neoplastic cells, epidermal cells, endodermal cells, smooth musclecells, among others.

As used herein, the language "vitamin D₃ agonist" refers to a compoundwhich potentiates, induces or otherwise enhances a biological activityof vitamin D₃ in a responsive cell. In certain embodiments, an agonistmay induce a genomic activity, e.g., activation of transcription by avitamin D₃ nuclear receptor, or a non-genomic vitamin D₃ activity, e.g.,potentiation of calcium channel activity. In other embodiments, theagonist potentiates the sensitivity of the receptor to another vitaminD₃ compound, e.g., treatment with the agonist lowers the concentrationof vitamin D₃ compound required to induce a particular biologicalresponse. The language "vitamin D₃ antagonist" is intended to includethose compounds that oppose any biological activity of a vitamin D₃compound.

The language "non-genomic" vitamin D₃ activities include cellular (e.g.,calcium transport across a tissue) and subcellular activities (e.g.,membrane calcium transport opening of voltage-gated calcium channels,changes in intracellular second messengers) elicited by vitamin D₃compounds in a responsive cell. Electrophysiological and biochemicaltechniques for detecting these activities are known in the art. Anexample of a particular well-studied non-genomic activity is the rapidhormonal stimulation of intestinal calcium mobilization, termed"transcaltachia" (Nemere I. et al. (1984) Endocrinology 115:1476-1483;Lieberherr M. et al. (1989) J. Biol. Chem. 264:20403-20406; Wali R. K.et al. (1992) Endocrinology 131:1125-1133; Wali R. K. et al. (1992) Am.J. Physiol. 262:G945-G953; Wali R. K. et al. (1990) J. Clin. Invest.85:1296-1303; Bolt M. J. G. et al. (1993) Biochem. J. 292:271-276).Detailed descriptions of experimental transcaltachia are provided inNorman, A. W. (1993) Endocrinology 268(27):20022-20030; Yoshimoto, Y.and Norman, A. W. (1986) Endocrinologyl 18:2300-2304. Changes in calciumactivity and second messenger systems are well known in the art and areextensively reviewed in Bouillion, R. et al. (1995) Endocrinology Review16(2): 200-257; the description of which is incorporated herein byreference.

Exemplary systems and assays for testing non-genomic activity areextensively described in the following references, liver (Baran D. T. etal. (1989) FEBS Lett 259:205-208; Baran D. T. et al. (1990) J. BoneMiner Res. 5:517-524;; rat osteoblasts, e.g., ROS 17/2.8 cells (Baran D.T. et al. (1991) J. Bone Miner Res. 6:1269-1275; Caffrey J. M. (1989) J.Biol. Chem. 264:20265-20274; Civitelli R. et al. (1990) Endocrinology127:2253-2262), muscle (DeBoland A. R. and Boland R. L. (1993) Biochem.Biophys Acta Mol. Cell Res. 1179:93-104; Morelli S. et al. (1993)Biochem J. 289:675-679; Selles J. and Boland R. L. (1991) Mol. CellEndocrinol. 82:229-235), and in parathyroid cells (Bourdeau A. et al.(1990) Endocrinology 127:2738-2743).

The language "genomic" activities or effects of vitamin D₃ is intendedto include those activities mediated by the nuclear/cytosol receptor for1α,25(OH)₂ D₃ (VD3R), e.g., transcriptional activation of target genes.The term "VD3Rs" is intended to include members of the type II class ofsteroid/thyroid superfamily of receptors (Stunnenberg, H. G. (1993) BioEssays 15(5):309-15), which are able to bind transactivate through thevitamin D response element (VDRE) in the absence of a ligand (Damm etal. (1989) Nature 339:593-97; Sap et al. Nature 343:177-180). As usedherein "VDREs" refer to a DNA sequences composed of half-sites arrangedas direct repeats. It is known in the art that type II receptors do notbind to their respective binding site as homodimers but require anauxiliary factor, RXR (e.g. RXRα, RXRβ, RXRγ) for high affinity bindingYu et al. (1991) Cell 67:1251-1266; Bugge et al. (1992) EMBO J.11:1409-1418; Kliewer et al. (1992) Nature 355:446-449; Leid et al.(1992) EMBO J. 11:1419-1435; Zhang et al. (1992) Nature 355:441-446).

Following binding, the transcriptional activity of a target gene (i.e.,a gene associated with the specific DNA sequence) is enhanced as afunction of the ligand bound to the receptor heterodimer. Exemplaryvitamin D₃ -responsive genes include osteocalcin, osteopontin,calbindins, parathyroid hormone (PTH), 24-hydroxylase, and α_(v) β₃-integrin. Genomic activities elicited by vitamin D3 compounds can betested by detecting the transcriptional upregulation of a vitamin D₃responsive gene in a cell containing VD3R_(S). For example, the steadystate levels of responsive gene mRNA or protein, e.g. calbindin gene,osteocalcin gene, can be detected in vivo or in vitro. Suitable cellsthat can be used include any vitamin D3-responsive cell, e.g.,keratinocytes, parathyroid cells, MG-63 cell line, among others.

In accordance with a still further embodiment of the present invention,convenient screening methods can be established in cell lines containingVD₃ R_(S), comprising (i) establishing a culture of these cells whichinclude a reporter gene construct having a reporter gene which isexpressed in an VD₃ R-dependent fashion; (ii) contacting these cellswith vitamin D3 compounds of formulas I and II; and (iii) monitoring theamount of expression of the reporter gene. Expression of the reportergene reflects transcriptional activity of the VD₃ R_(S) protein.Typically, the reporter gene construct will include a reporter gene inoperative linkage with one or more transcriptional regulatory elementsresponsive to VD₃ R_(S), e.g., the VD₃ R_(S) response element (VDRE)known in the art. The amount of transcription from the reporter gene maybe measured using any method known to those of skill in the art to besuitable. For example, specific mRNA expression may be detected usingNorthern blots or specific protein product may be identified by acharacteristic stain, immunoassay or an intrinsic activity. In preferredembodiments, the gene product of the reporter is detected by anintrinsic activity associated with that product. For instance, thereporter gene may encode a gene product that, by enzymatic activity,gives rise to a detection signal based on color, fluorescence, orluminescence. The amount of expression from the reporter gene is thencompared to the amount of expression in either the same cell in theabsence of the test compound or it may be compared with the amount oftranscription in a substantially identical cell that lacks the specificreceptors. Agonistic vitamin D₃ compounds can then be readily detectedby the increased activity or concentration of these reporter genesrelative to untransfected controls.

After identifying certain test compounds as potential agonists orantagonists of vitamin D₃ compounds, the practioner of the subject assaywill continue to test the efficacy and specificity of the selectedcompounds both in vitro and in vivo. Whether for subsequent in vivotesting, or for administration to an animal as an approved drug, agentsidentified in the subject assay can be formulated in pharmaceuticalpreparations, such as described above, for in vivo administration to ananimal, preferably a human.

As described herein, the vitamin D3 compounds of the present inventionshow improved biological properties than vitamin D3. As used herein, thelanguage "improved biological properties" refers to any activityinherent in a vitamin D3 compound of formula I or II that enhances itseffectiveness in vivo. In a preferred embodiment, this term refers toany qualitative or quantitative improved therapeutic property of avitamin D₃ compound, such as enhanced stability in vivo and/or reducedtoxicity, e.g., reduced hypercalcemic activity. The improved biologicalproperty may occur in both a tissue-specific and non-specific manner.For example, certain tissues may be capable of metabolizing vitamin D₃into unique metabolites that enhance in a tissue-specific manner thebiological activities of this compound.

The increased stability of the vitamin D3 compounds of formulas I and IIcan be demonstrated in incubation studies, wherein a significantlyhigher concentration of the such vitamin D3 after prolonged incubationsin vivo or in vitro, or an increase in the binding to plasma vitamin Dbinding protein (DBP) compared to vitamin D3 indicates a compound havingenhanced stability (See A. W. Norman et al. J. Biol. Chem. 268 (27):20022-20030).

The language "reduced toxicity" is intended to include a reduction inany undesired side effect elicited by a vitamin D3 compound of formula Ior II when administered in vivo, e.g., a reduction in the hypercalcemicactivity. The language "hypercalcemia" or "hypercalcemic activity" isintended to have its accepted clinical meaning, namely, increases incalcium serum levels that are manifested in a subject by the followingside effects, depression of central and peripheral nervous system,muscular weakness, constipation, abdominal pain, lack of appetite and,depressed relaxation of the heart during diastole. Symptomaticmanifestations of hypercalcemia are triggered by a stimulation of atleast one of the following activities, intestinal calcium transport,bone calcium metabolism and osteocalcin synthesis (reviewed in Boullion,R. et al. (1995) Endocrinology Reviews 16(2): 200-257).

Compounds exhibiting reduced hypercalcemic activity can be tested invivo or in vitro using methods known in the art and reviewed byBoullion, R. et al. (1995) Endocrinology Reviews 16(2): 200-257. Forexample, the serum calcium levels following administration of a vitaminD3 compounds of formula I or II can be tested by routine experimentation(Lemire, J. M. (1994) Endocrinology 135(6):2818-2821). Briefly, vitaminD3 compounds of formulas I and II can be administered intramuscularly tovitamin D₃ -deficient subjects, e.g., rodents, e.g. mouse, or avianspecies, e.g. chick. At appropriate time intervals, serum calcium levelsand extent of calcium uptake can be used to determine the level of bonecalcium mobilization (BCM) and intestinal calcium absorption (ICA)induced by the tested vitamin D₃ compound as described in Norman, A. W.et al. (1993) J. Biol. Chem. 268(27):20022-20029. Compounds which uponaddition fail to increase the concentration of calcium in the bloodserum, thus showing decreased BCM and ICA responses compared to theirisomeric counterparts, are considered to have reduced hypercalcemicactivity. Compounds which have reduced toxicity compared to theirisomeric counterparts are considered to have reduced toxicity.Additional calcium homeostasis-related assays are described below in theCalcium and Phosphate Homeostasis section.

Hyperproliferative Conditions

In another aspect the present invention provides a method of treating ina subject, a disorder characterized by aberrant activity of a vitaminD3-responsive cell. The method involves administering to the subject aneffective amount of a pharmaceutical composition of a vitamin D3compound of formula I or II such that the activity of the cell ismodulated. As used herein, the language "modulate" refers to increasesor decreases in the activity of a cell in response to exposure to acompound of the invention, e.g., the inhibition of proliferation and/orinduction of differentiation of at least a sub-population of cells in ananimal such that a desired end result is achieved, e.g. a therapeuticresult. In preferred embodiments, this phrase is intended to includehyperactive conditions that result in pathological disorders.

In certain embodiments, the cells to be treated are hyperproliferativecells. As described in greater detail below, the vitamin D3 compounds offormulas I and II can be used to inhibit the proliferation of a varietyof hyperplastic and neoplastic tissues. In accordance with the presentinvention, vitamin D3 compounds of formulas I and II can be used in thetreatment of both pathologic and non-pathologic proliferative conditionscharacterized by unwanted growth of vitamin D3-responsive cells, e.g.,hyperproliferative skin cells, immune cells, and tissue havingtransformed cells, e.g., such as carcinomas, sarcomas and leukemias. Inother embodiments, the cells to be treated are aberrant secretory cells,e.g., parathyroid cells, immune cells.

As used herein, the terms "hyperproliferative" and "neoplastic" are usedinterchangeably, and include those cells having the capacity forautonomous growth, i.e., an abnormal state or condition characterized byrapidly proliferating cell growth. Hyperproliferative and neoplasticdisease states may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e.. a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. "Pathologic hyperproliferative" cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The use of vitamin D3 compounds of formulas I and II in treatinghyperproliferative conditions has been limited because of theirhypercalcemic effects. Thus, vitamin D3 compounds of formula I and IIcan provide a less toxic alternative to current methods of treatment.

In one embodiment, this invention features a method for inhibiting theproliferation and/or inducing the differentiation of ahyperproliferative skin cell, e.g., an epidermal or an epithelial cell,e.g. a keratinocytes, by contacting the cells with a vitamin D3 compoundof formula I or II. In general, the method includes a step of contactinga pathological or non-pathological hyperproliferative cell with aneffective amount of such vitamin D3 compound to promote thedifferentiation of the hyperproliferative cells The present method canbe performed on cells in culture, e.g. in vitro or ex vivo, or can beperformed on cells present in an animal subject, e.g., as part of an invivo therapeutic protocol. The therapeutic regimen can be carried out ona human or any other animal subject.

The vitamin D3 compounds of the present invention can be used to treat ahyperproliferative skin disorder. Exemplary disorders include, but arenot limited to, psoriasis, basal cell carcinoma, keratinizationdisorders and keratosis. Additional examples of these disorders includeeczema; lupus associated skin lesions; psoriatic arthritis; rheumatoidarthritis that involves hyperproliferation and inflammation ofepithelial-related cells lining the joint capsule; dermatitides such asseborrheic dermatitis and solar dermatitis; keratoses such as seborrheickeratosis, senile keratosis, actinic keratosis. photo-induced keratosis,and keratosis follicularis; acne vulgaris; keloids and prophylaxisagainst keloid formation; nevi; warts including verruca, condyloma orcondyloma acuminatum, and human papilloma viral (HPV) infections such asvenereal warts; leukoplakia; lichen planus; and keratitis.

In an illustrative example, vitamin D3 compounds of formulas I and IIcan be used to inhibit the hyperproliferation of keratinocytes intreating diseases such as psoriasis by administering an effective amountof these compounds to a subject in need of treatment. The term"psoriasis" is intended to have its medical meaning, namely, a diseasewhich afflicts primarily the skin and produces raised, thickened,scaling, nonscarring lesions. The lesions are usually sharply demarcatederythematous papules covered with overlapping shiny scales. The scalesare typically silvery or slightly opalescent. Involvement of the nailsfrequently occurs resulting in pitting, separation of the nail,thickening and discoloration. Psoriasis is sometimes associated witharthritis, and it may be crippling. Hyperproliferation of keratinocytesis a key feature of psoriatic epidermal hyperplasia along with epidermalinflammation and reduced differentiation of keratinocytes. Multiplemechanisms have been invoked to explain the keratinocytehyperproliferation that characterizes psoriasis. Disordered cellularimmunity has also been implicated in the pathogenesis of psoriasis.

Pharmaceutical compositions of vitamin D3 compounds of formulas I and IIcan be delivered or administered topically or by transdermal patches fortreating dermal psoriasis. Alternatively, oral administration is used.Additionally, the compositions can be delivered parenterally, especiallyfor treatment of arthritis, such as psoriatic arthritis, and for directinjection of skin lesions. Parenteral therapy is typically intra-dermal,intra-articular, intramuscular or intravenous. A preferred way topractice the invention is to apply the vitamin D3 compounds of formulasI and II, in a cream or oil based carrier, directly to the psoriaticlesions. Typically, the concentration of the vitamin D3 compound in acream or oil is 1-2%. Alternatively, an aerosol can be used topically.These compounds can also be orally administered.

In general, the route of administration is topical (includingadministration to the eye, scalp, and mucous membranes), oral, orparenteral. Topical administration is preferred in treatment of skinlesions, including lesions of the scalp, lesions of the cornea(keratitis), and lesions of mucous membranes where such directapplication is practical. Shampoo formulations are sometimesadvantageous for treating scalp lesions such as seborrheic dermatitisand psoriasis of the scalp. Mouthwash and oral paste formulations can beadvantageous for mucous membrane lesions, such as oral lesions andleukoplakia. Oral administration is a preferred alternative fortreatment of skin lesions and other lesions discussed above where directtopical application is not as practical, and it is a preferred route forother applications.

Intra-articular injection is a preferred alternative in the case oftreating one or only a few (such as 2-6) joints. Additionally, thetherapeutic compounds are injected directly into lesions (intra-lesionadministration) in appropriate cases. Intra-dermal administration is analternative for dermal lesions such as those of psoriasis.

The amount of the pharmaceutical composition to be administered variesdepending upon the type of the disease of a patient, the severity of thedisease, the type of the active vitamin D₃ compound of Formulas I or II,among others. For example, the vitamin D3 compound of formula I or IIcan be administered topically for treating hyperproliferative skinconditions at a dose in the range of 1 to 1000 μg per gram of topicalformulation.

Neoplasia

Another embodiment features methods for inhibiting the proliferationand/or reversing the transformed phenotype of vitamin D3-responsivehyperproliferative cells by contacting the cells with a vitamin D3compound of formula I or II. In general, the method includes a step ofcontacting pathological or non-pathological hyperproliferative cellswith an effective amount of a vitamin D3 compound of formula I or II forpromoting the differentiation of the hyperproliferative cells. Thepresent method can be performed on cells in culture, e.g., in vitro orex vivo, or can be performed on cells present in an animal subject,e.g., as part of an in vivo therapeutic protocol. The therapeuticregimen can be carried out on a human or other animal subject.

The terms "antineoplastic agent" and "antiproliferative agent" are usedinterchangeably herein and includes agents that have the functionalproperty of inhibiting the proliferation of a vitamin D3-responsivecells, e.g., inhibit the development or progression of a neoplasm havingsuch a characteristic, particularly a hematopoietic neoplasm.

As used herein, a "therapeutically effective anti-neoplastic amount" ofa vitamin D3 compound of formula I or II refers to an amount of an agentwhich is effective, upon single or multiple dose administration to thepatient, in inhibiting the growth of a neoplastic vitamin D3-responsivecells, or in prolonging the survivability of the patient with suchneoplastic cells beyond that expected in the absence of such treatment.As used herein, "inhibiting the growth" of the neoplasm includes theslowing, interrupting, arresting or stopping its growth and metastasesand does not necessarily indicate a total elimination of the neoplasticgrowth.

As used herein, "a prophylactically effective anti-neoplastic amount" ofa compound refers to an amount of a vitamin D3 compound of formula I orII which is effective, upon single or multiple dose administration tothe patient, in preventing or delaying the occurrence of the onset of aneoplastic disease state.

The common medical meaning of the term "neoplasia" refers to "new cellgrowth" that results as a loss of responsiveness to normal growthcontrols, e.g. to neoplastic cell growth. A "hyperplasia" refers tocells undergoing an abnormally high rate of growth. However, as usedherein, the terms neoplasia and hyperplasia can be used interchangably,as their context will reveal, referring to generally to cellsexperiencing abnormal cell growth rates. Neoplasias and hyperplasiasinclude "tumors," which may be either benign, premalignant or malignant.

The vitamin D3 compounds of formulas I and II can be tested initially invitro for their inhibitory effects in the proliferation of neoplasticcells. Examples of cell lines that can be used are transformed cells,e.g., the human promyeloid leukemia cell line HL-60, and the humanmyeloid leukemia U-937 cell line (Abe E. et al. (1981) Proc. Natl. Acad.Sci. USA 78:4990-4994; Song L. N. and Cheng T. (1992) Biochem Pharmacol43:2292-2295; Zhou J. Y. et al. (1989) Blood 74:82-93; U.S. Pat. Nos.5,401,733, 5,087,619). Alternatively, the antitumoral effects of vitaminD3 compounds of formulas I and II can be tested in vivo using variousanimal models known in the art and summarized in Bouillon, R. et al.(1995) Endocrine Reviews 16(2):233 (Table E), which is incorporated byreference herein. For example, SL mice are routinely used in the art totest vitamin D3 compounds of formulas I and II as models for MI myeloidleukemia (Honma et al. (1983) Cell Biol. 80:201-204; Kasukabe T. et al.(1987) Cancer Res. 47:567-572); breast cancer studies can be performedin, for example, nude mice models for human MX1 (ER) (Abe J. et al.(1991) Endocrinology 129:832-837; other cancers, e.g., colon cancer,melanoma osteosarcoma, can be characterized in, for example, nude micemodels as describe in (Eisman J. A. et al. (1987) Cancer Res. 47:21-25;Kawaura A. et al. (1990) Cancer Lett 55:149-152; Belleli A. (1992)Carcinogenesis 13:2293-2298; Tsuchiya H. et al. (1993) J. Orthopaed Res.11:122-130).

The subject method may also be used to inhibit the proliferation ofhyperplastic/neoplastic cells of hematopoietic origin, e.g., arisingfrom myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. For instance, the present invention contemplates the treatmentof various myeloid disorders including, but not limited to, acutepromyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97). Lymphoid malignancies which may be treatedby the subject method include, but are not limited to acutelymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas contemplated by the treatmentmethod of the present invention include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF) and Hodgkin's disease.

The term "leukemia" is intended to have its clinical meaning, namely, aneoplastic disease in which white corpuscle maturation is arrested at aprimitive stage of cell development. The disease is characterized by anincreased number of leukemic blast cells in the bone marrow, and byvarying degrees of failure to produce normal hematopoietic cells. Thecondition may be either acute or chronic. Leukemias are furthertypically categorized as being either lymphocytic i.e., beingcharacterized by cells which have properties in common with normallymphocytes, or myelocytic (or myelogenous), i.e., characterized bycells having some characteristics of normal granulocytic cells. Acutelymphocytic leukemia ("ALL") arises in lymphoid tissue, and ordinarilyfirst manifests its presence in bone marrow. Acute myelocytic leukemia("AML") arises from bone marrow hematopoietic stem cells or theirprogeny. The term acute myelocytic leukemia subsumes several subtypes ofleukemia: myeloblastic leukemia, promyelocytic leukemia, andmyelomonocytic leukemia. In addition, leukemias with erythroid ormegakaryocytic properties are considered myelogenous leukemias as well.

As used herein the term "leukemic cancer" refers to all cancers orneoplasias of the hemopoietic and immune systems (blood and lymphaticsystem). The acute and chronic leukemias, together with the other typesof tumors of the blood, bone marrow cells (myelomas), and lymph tissue(lymphomas), cause about 10% of all cancer deaths and about 50% of allcancer deaths in children and adults less than 30 years old. Chronicmyelogenous leukemia (CML), also known as chronic granulocytic leukemia(CGL), is a neoplastic disorder of the hematopoietic stem cell. The term"leukemia" is art recognized and refers to a progressive, malignantdisease of the blood-forming organs, marked by distorted proliferationand development of leukocytes and their precursors in the blood and bonemarrow.

In certain embodiments, the vitamin D3 compounds of formulas I and IIcan be used in combinatorial therapy with conventional cancerchemotherapeutics. Conventional treatment regimens for leukemia and forother tumors include radiation, drugs, or a combination of both. Inaddition to radiation, the following drugs, usually in combinations witheach other, are often used to treat acute leukemias: vincristine,prednisone, methotrexate, mercaptopurine, cyclophosphamide, andcytarabine. In chronic leukemia, for example, busulfan, melphalan, andchlorambucil can be used in combination. All of the conventionalanti-cancer drugs are highly toxic and tend to make patients quite illwhile undergoing treatment. Vigorous therapy is based on the premisethat unless every leukemic cell is destroyed, the residual cells willmultiply and cause a relapse.

The subject method can also be useful in treating malignancies of thevarious organ systems, such as affecting lung, breast, lymphoid,gastrointestinal, and genitourinary tract as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

The term "carcinoma" is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An "adenocarcinoma" refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term "sarcoma" is art recognized and refers to malignant tumors ofmesenchymal derivation.

According to the general paradigm of vitamin D3 involvement indifferentiation of transformed cells, exemplary solid tumors that can betreated according to the method of the present invention include vitaminD3-responsive phenotypes of sarcomas and carcinomas such as, but notlimited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Determination of a therapeutically effective anti-neoplastic amount or aprophylactically effective anti-neoplastic amount of the vitamin D3compound of formula I or II, can be readily made by the physician orveterinarian (the "attending clinician"), as one skilled in the art, bythe use of known techniques and by observing results obtained underanalogous circumstances. The dosages may be varied depending upon therequirements of the patient in the judgment of the attending clinician,the severity of the condition being treated and the particular compoundbeing employed. In determining the therapeutically effectiveantineoplastic amount or dose, and the prophylactically effectiveantineoplastic amount or dose, a number of factors are considered by theattending clinician, including, but not limited to: the specifichyperplastic/neoplastic cell involved; pharmacodynamic characteristicsof the particular agent and its mode and route of administration; thedesirder time course of treatment; the species of mammal; its size, age,and general health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the kind of concurrenttreatment (i.e., the interaction of the vitamin D3 compounds of formulasI and II with other co-administered therapeutics); and other relevantcircumstances. U.S. Pat. No. 5,427,916, for example, describes methodfor predicting the effectiveness of antineoplastic therapy in individualpatients, and illustrates certain methods which can be used inconjunction with the treatment protocols of the instant invention.

Treatment can be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage should be increasedby small increments until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired. A therapeuticallyeffective antineoplastic amount and a prophylactically effectiveanti-neoplastic amount of a vitamin D3 compound of formula I or II isexpected to vary from about 0.1 milligram per kilogram of body weightper day (mg/kg/day) to about 100 mg/kg/day.

Compounds which are determined to be effective for the prevention ortreatment of tumors in animals, e.g., dogs, rodents, may also be usefulin treatment of tumors in humans. Those skilled in the art of treatingtumor in humans will know, based upon the data obtained in animalstudies, the dosage and route of administration of the compound tohumans. In general, the dosage and route of administration in humans isexpected to be similar to that in animals.

The identification of those patients who are in need of prophylactictreatment for hyperplastic/neoplastic disease states is well within theability and knowledge of one skilled in the art. Certain of the methodsfor identification of patients which are at risk of developingneoplastic disease states which can be treated by the subject method areappreciated in the medical arts, such as family history of thedevelopment of a particular disease state and the presence of riskfactors associated with the development of that disease state in thesubject patient. The present application also describes other prognostictests which can be used to make, or to augment a clinical predicationabout the use of the method of the present invention. A clinicianskilled in the art can readily identify such candidate patients, by theuse of, for example, clinical tests, physical examination andmedical/family history.

Immunomodulatory Effects

In another aspect, this invention provides a method for modulating theactivity of an immune cell by contacting the cell with a vitamin D3compound of formulas I or II. Vitamin D3 compounds are known in the artfor their inhibitory effects on the antigen-specific immune system. Asused herein, the phrase "inhibition of an immune response" is intendedto include decreases in T cell proliferation and activity, e.g., adecrease in IL₂, interferon-γ, GM-CSF synthesis and secretion (Lemire,J. M. (1992) J. Cell Biochemistry 49:26-31, Lemire, J. M. et al. (1994)Endocrinology 135 (6): 2813-2821; Bouillon, R. et al. (1995) EndocineReview 16 (2):231-32)

In one embodiment, the present invention provides a method forsuppressing immune activity in an immune cell by contacting apathological or non-pathological immune cell with an effective amount ofa vitamin D3 compound of formulas I or II to thereby inhibit an immuneresponse relative to the cell in the absence of the treatment. Thepresent method can be performed on cells in culture, e.g., in vitro orex vivo, or can be performed on cells present in an animal subject,e.g., as part of an in vivo therepeutic protocol. In vivo treatment canbe carried out on a human or other animal subject.

The vitamin D3 compound of formula I or II can be tested initially invitro for their inhibitory effects on T cell proliferation and secretoryactivity, as described in Reichel, H. et al., (1987) Proc. Natl. Acad.Sci. USA 84:3385-3389; Lemire, J. M. et al. (1985) J. Immunol34:2032-2035. Alternatively, the immunosuppressive effects can be testedin vivo using the various animal models known in the art and summarizedby Bouillon, R. et al. (1995) Endocine Reviews 16(2) 232 (Tables 6 and7). For examples, animal models for autoimmune disorders, e.g., lupus,thyroiditis, encephalitis, diabetes and nephritis are described in(Lemire J. M. (1992). J. Cell Biochem. 49:26-31; Koizumi T. et al.(1985) Int. Arch. Allergy Appl. Immunol. 77:396-404; Abe J. et al.(1990) Calcium Regulation and Bone Metabolism 146-151; Fournier C. etal. (1990) Clin. Immunol Immunopathol. 54:53-63; Lemire J. M. and ArcherD. C. (1991) J. Clin. Invest. 87:1103-1107); Lemire, J. M. et al.,(1994) Endocrinology 135 (6):2818-2821; Inaba M. et al. (1992)Metabolism 41:631-635; Mathieu C. et al. (1992) Diabetes 41:1491-1495;Mathieu C. et al. (1994) Diabetologia 37:552-558; Lillevang S. T. et al.(1992) Clin. Exp. Immunol. 88:301-306, among others). Models forcharacterizing immunosuppressuve activity during organ transplantation,e.g., skin graft, cardiac graft, islet graft, are described in Jordan S.C. et al. (1988) v Herrath D (eds) Molecular, Cellular and ClinicalEndocrinology 346-347; Veyron P. et al. (1993) Transplant Immunol.1:72-76; Jordan S. C. (1988) v Herrath D (eds) Molecular, Cellular andClinical Endocrinology 334-335; Lemire J. M. et al. (1992)Transplantation 54:762-763; Mathieu C. et al. (1994) Transplant Proc.26:3128-3129).

After identifying certain test compounds as effective suppresors of animmune response in vitro, these compounds can be used in vivo as part ofa therapeutic protocol. Accordingly, another embodiment provides amethod of suppressing an immune response, comprising administering to asubject a pharmaceutical preparation of a vitamin D3 compound of formulaI or II, so as to inhibit immune reactions such as graft rejection,autoimmune disorders and inflammation.

For example, the subject vitamin D3 compounds of formulas I and II canbe used to inhibit responses in clinical situations where it isdesirable to downmodulate T cell responses. For example, ingraft-versus-host disease, cases of transplantation, autoimmune diseases(including, for example, diabetes mellitus, arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), multiple sclerosis, encephalomyelitis, diabetes,myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis,dermatitis (including atopic dermatitis and eczematous dermatitis),psoriasis, Sjogren's Syndrome, including keratoconjunctivitis siccasecondary to Sjogren's Syndrome, alopecia areata, allergic responses dueto arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, and interstitial lungfibrosis). Downmodulation of immune activity will also be desirable incases of allergy such as, atopic allergy.

As described before, determination of a therapeutically effectiveimmunosuppressive amount can be readily made by the attending clinician,as one skilled in the art, by the use of known techniques and byobserving results obtained under analogous circumstances. Compoundswhich are determined to be effective in animals, e.g., dogs, rodents,may be extrapolated accordingly to humans by those skilled in the art.Starting dose/regimen used in animals can be estimated based on priorstudies. For example, doses of vitamin D3 compounds of formulas I and IIto treat autoimmune disorders in rodents can be initially estimated inthe range of 0.1 g/kg/day to 1 g/kg/day, administered orally or byinjection.

Those skilled in the art will know based upon the data obtained inanimal studies, the dosage and route of administration in humans isexpected to be similar to that in animals. Exemplary dose ranges to beused in humans are from 0.25 to 10 μg/day, preferably 0.5 to 5 μg/dayper adult (U.S. Pat. No. 4,341,774).

Calcium and Phosphate Homeostasis

The present invention also relates to a method of treating in a subjecta disorder characterized by deregulation of calcium metabolism. Thismethod comprises contacting a pathological or non-pathological vitaminD3 responsive cell with an effective amount of a vitamin D3 compound offormula I or II to thereby directly or indirectly modulate calcium andphosphate homeostasis. The term "homeostasis" is art-recognized to meanmaintenance of static, or constant, conditions in an internalenvironment. As used herein, the term "calcium and phospate homeostasis"refers to the careful balance of calcium and phosphate concentrations,intracellularly and extracellularly, triggered by fluctuations in thecalcium and phosphate concentration in a cell, a tissue, an organ or asystem. Fluctuations in calcium levels that result from direct orindirect responses to vitamin D3 compounds of formulas I and II areintended to be included by these terms. Techniques for detecting calciumfluctuation in vivo or in vitro are known in the art.

Exemplary Ca⁺⁺ homeostasis related assays include assays that focus onthe intestine where intestinal ⁴⁵ Ca²⁺ absorption is determinedeither 1) in vivo (Hibberd K. A. and Norman A. W. (1969) Biochem.Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967) J. Nutr. 91:319-323;Bickle D. D. et al. (1984) Endocrinology 114:260-267), or 2) in vitrowith everted duodenal sacs (Schachter D. et al. (1961) Am. J. Physiol200:1263-1271), or 3) on the genomic induction of calbindin-D_(28k) inthe chick or of calbindin-D_(9k) in the rat (Thomasset M. et al. (1981)FEBS Lett. 127:13-16; Brehier A. and Thomasset M. (1990) Endocrinology127:580-587). The bone-oriented assays include: 1) assessment of boneresorption as determined via the release of Ca²⁺ from bone in vivo (inanimals fed a zero Ca²⁺ diet) (Hibberd K. A. and Norman A. W. (1969)Biochem. Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967) J. Nutr.91:319-323), or from bone explants in vitro (Bouillon R. et al. (1992)J. Biol. Chem. 267:3044-3051), 2) measurement of serum osteocalcinlevels [osteocalcin is an osteoblast-specific protein that after itssynthesis is largely incorporated into the bone matrix, but partiallyreleased into the circulation (or tissue culture medium) and thusrepresents a good market of bone formation or turnover] (Bouillon R. etal. (1992) Clin. Chem. 38:2055-2060), or 3) bone ash content (Norman A.W. and Wong R. G. (1972) J. Nutr. 102:1709-1718). Only onekidney-oriented assay has been employed. In this assay, urinary Ca²⁺excretion is determined (Hartenbower D. L. et al. (1977) Walter deGruyter, Berlin pp 587-589); this assay is dependent upon elevations inthe serum Ca²⁺ level and may reflect bone Ca²⁺ mobilizing activity morethan renal effects. Finally, there is a "soft tissue calcification"assay that has been employed to detect the consequences of 1α,25(OH)₂ D3or analog-induced severe hypercalcemia. In this assay a rat isadministered an intraperitoneal dose of ⁴⁵ Ca²⁺, followed by seven dailyrelative high doses of 1α,25(OH)₂ D3 or the analog of interest; in theevent of onset of a severe hypercalcemia, soft tissue calcification canbe assessed by determination of the ⁴⁵ Ca²⁺ level. In all these assays,vitamin D3 compounds or formulas I and II are administered to vitaminD-sufficient or -deficient animals, as a single dose or chronically(depending upon the assay protocol), at an appropriate time intervalbefore the end point of the assay is quantified.

In certain embodiments, vitamin D3 compounds of formulas I and II can beused to modulate bone metabolism. The language "bone metabolism" isintended to include direct or indirect effects in the formation ordegeneration of bone structures, e.g., bone formation, bone resorption,etc., which may ultimately affect the concentrations in serum of calciumand phosphate. This term is also intended to include effects of vitaminD3 compounds in bone cells, e.g. osteoclasts and osteoblasts, that mayin turn result in bone formation and degeneration. For example, it isknown in the art, that vitamin D3 compounds of formulas I and II exerteffects on the bone forming cells, the osteoblasts through genomic andnon-genomic pathways (Walters M. R. et al. (1982) J. Biol. Chem.257:7481-7484; Jurutka P. W. et al. (1993) Biochemistry 32:8184-8192;Mellon W. S. and DeLuca H. F. (1980) J. Biol. Chem. 255:4081-4086).Similarly, vitamin D3 compounds of formulas I and II are known in theart to support different activities of bone resorbing osteoclasts suchas the stimulation of differentiation of monocytes and mononuclearphagocytes into osteoclasts (Abe E. et al. (1988) J. Bone Miner Res.3:635-645; Takahashi N. et al. (1988) Endocrinology 123:1504-1510;Udagawa N. et al. (1990) Proc. Natl. Acad. Sci. USA 87:7260-7264).Accordingly, vitamin D3 compounds of formulas I and II that modulate theproduction of bone cells can influence bone formation and degeneration.

The present invention provides a method for modulating bone cellmetabolism by contacting a pathological or a non-pathological bone cellwith an effective amount of a vitamin D3 compound of formula I or II tothereby modulate bone formation and degeneration. The present method canbe performed on cells in culture, e.g., in vitro or ex vivo, or can beperformed in cells present in an animal subject, e.g., cells in vivo.Exemplary culture systems that can be used include osteoblast celllines, e.g., ROS 17/2.8 cell line, monocytes, bone marrow culture system(Suda T. et al. (1990) Med. Res. Rev. 7:333-366; Suda T. et al. (1992)J. Cell Biochem. 49:53-58) among others. Selected compounds can befurther tested in vivo, for example, animal models of osteopetrosis andin human disease (Shapira F. (1993) Clin. Orthop. 294:34-44).

In a preferred embodiment, a method for treating osteoporosis isprovided, comprising administering to a subject a pharmaceuticalpreparation of a vitamin D3 compound of formula I or II to therebyameliorate the condition relative to an untreated subject. The rationalefor utilizing vitamin D3 compounds in the treatment of osteoporosis issupported by studies indicating a decrease in serum concentration of1α,25(OH)₂ D3 in elderly subjects (Lidor C. et al. (1993) Calcif. TissueInt. 52:146-148). In vivo studies using vitamin D3 compounds in animalmodels and humans are described in Bouillon, et al. (1995) EndocrineReviews 16(2):229-231.

Vitamin D3 compounds of formulas I and II can be tested in ovarectomizedanimals, e.g., dogs, rodents, to assess the changes in bone mass andbone formation rates in both normal and estrogen-deficient animals.Clinical trials can be conducted in humans by attending clinicians todetermine therapeutically effective amounts of the vitamin D3 compoundsof formulas I and II in preventing and treating osteoporosis.

Preferred compounds to be tested include 3-epi forms of 3-epi forms of1α(OH)D₃ as shown in Example II, which shows the production of1α(OH)-3-epi-D₃ in the rat osteosarcoma cell line UMR-106. The 3 epiconversion of 1α(OH)-D₃ presents the possibility of a yet improved ofthis compound.

In other embodiments, therapeutic applications of the vitamin D3compounds of formulas I and II include treatment of other diseasescharacterized by metabolic calcium and phosphate deficiencies. Exemplaryof such diseases are the following: osteoporosis, osteodystrophy,osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy,osteosclerosis, anti-convulsant treatment, osteopenia,fibrogenesis-imperfecta ossium, secondary hyperparathyrodism,hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructivejaundice, drug induced metabolism, medullary carcinoma, chronic renaldisease, hypophosphatemic VDRR, vitamin D-dependent rickets,sarcoidosis, glucocorticoid antagonism, malabsorption syndrome,steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

Hormone Secretion

In yet another aspect, the present invention provides a method formodulating hormone secretion of a vitamin D3-responsive cell, e.g., anendocrine cell. The language "hormone secretion" is art-recognized andincludes both genomic and non-genomic activities of vitamin D3 compoundsof formulas I and II that control the transcription and processingresponsible for secretion of a given hormone e.g., parathyroid hormone(PTH), calcitonin, insulin, prolactin (PRL) and TRH in a vitamin D3responsive cell (Bouillon, R. et al. (1995) Endocrine Reviews16(2):235-237). The language "vitamin D3 responsive cells" as usedherein is intended to include endocrine cells which respond to vitaminD3 compounds of formulas I and II by altering gene expression and/orpost-transcriptional processing secretion of a hormone. Exemplaryendocrine cells include parathyroid cells, pancreatic cells, pituitarycells, among others.

The present method can be performed on cells in culture, e.g. in vitroor ex vivo, or on cells present in an animal subject, e.g., in vivo.Vitamin D3 compounds of formulas I and II can be initially tested invitro using primary cultures of parathyroid cells. Other systems thatcan be used include the testing by prolactin secretion in rat pituitarytumor cells, e.g., GH4C1 cell line (Wark J. D. and Tashjian Jr. A. H.(1982) Endocrinology 111:1755-1757; Wark J. D. and Tashjian Jr. A. H.(1983) J. Biol. Chem. 258:2118-2121; Wark J. D. and Gurtler V. (1986)Biochem. J. 233:513-518) and TRH secretion in GH4C1 cells.Alternatively, the effects of vitamin D3 compounds of formulas I and IIcan be characterized in vivo using animals models as described in Nko M.et al. (1982) Miner Electrolyte Metab. 5:67-75; Oberg F. et al. (1993)J. Immunol. 150:3487-3495; Bar-Shavit Z. et al. (1986) Endocrinology118:679-686; Testa U. et al. (1993) J. Immunol. 150:2418-2430; NakamakiT. et al. (1992) Anticancer Res. 12:1331-1337; Weinberg J. B. andLarrick J. W. (1987) Blood 70:994-1002; Chambaut-Guerin A. M. andThomopoulos P. (1991) Eur. Cytokine New. 2:355; Yoshida M. et al. (1992)Anticancer Res. 12:1947-1952; Momparler R. L. et al. (1993) Leukemia7:17-20; Eisman J. A. (1994) Kanis J A (eds) Bone and Mineral Research2:45-76; Veyron P. et al. (1993) Transplant Immunol. 1:72-76; Gross M.et al. (1986) J. Bone Miner Res. 1:457-467; Costa E. M. et al. (1985)Endocrinology 117:2203-2210; Koga M. et al. (1988) Cancer Res.48:2734-2739; Franceschi R. T. et al. (1994) J. Cell Physiol.123:401-409; Cross H. S. et al. (1993) Naunyn Schmiedebergs Arch.Pharmacol. 347:105-110; Zhao X. and Feldman D. (1993) Endocrinology132:1808-1814; Skowronski R. J. et al. (1993) Endocrinology132:1952-1960; Henry H. L. and Norman A. W. (1975) Biochem. Biophys.Res. Commun. 62:781-788; Wecksler W. R. et al. (1980) Arch. Biochem.Biophys. 201:95-103; Brumbaugh P. F. et al. (1975) Am. J. Physiol.238:384-388; Oldham S. B. et al. (1979) Endocrinology 104:248-254;Chertow B. S. et al. (1975) J. Clin Invest. 56:668-678; Canterbury J. M.et al. (1978) J. Clin. Invest. 61:1375-1383; Quesad J. M. et al. (1992)J. Chin. Endocrinol. Metab. 75:494-501.

In certain embodiments, the vitamin D3 compounds of the presentinvention can be used to inhibit parathyroid hormone (PTH) processing,e.g., transcriptional, translational processing, and/or secretion of aparathyroid cell as part of a therapeutic protocol. Therapeutic methodsusing these compounds can be readily applied to all diseases, involvingdirect or indirect effects of PTH activity, e.g., primary or secondaryresponses. For example, it is known in the art that PTH induces theformation of 1,25-dihydroxy vitamin D3 in the kidneys, which in turn inincreases calcium and phosphate absorption from the intestine thatcauses hypercalcemia. Thus inhibition of PTH processing and/or secretionwould indirectly inhibit all of the responses mediated by PTH in vivo.Accordingly, therapeutic applications for these vitamin D3 compounds offormulas I and II include treating diseases such as secondaryhyperparathyroidism of chronic renal failure (Slatopolsky E. et al.(1990) Kidney Int. 38:S41-S47; Brown A. J. et al. (1989) J. Clin.Invest. 84:728-732). Determination of therapeutically affective amountsand dose regimen can be performed by the skilled artisan using the datadescribed in the art.

Protection Against Neuronal Loss

In yet another aspect, the present invention provides a method ofprotecting against neuronal loss by contacting a vitamin D3 responsivecell, e.g., a neuronal cell, with a vitamin D3 compound of formula I orII to prevent or retard neuron loss. The language "protecting against"is intended to include prevention, retardation, and/or termination ofdeterioration, impairment, or death of a neurons. The language "vitaminD3 responsive cells" as used herein is intended to include neuronalcells which respond to vitamin D3 compounds of formulas I and II byaltering gene expression and/or intracellular metabolism. Exemplaryneuronal cells include hippocampal cells, dopaminergic cells,cholinergic cells, among others.

Neuron loss can be the result of any condition of a neuron in which itsnormal function is compromised. Neuron deterioration can be the resultof any condition which compromises neuron function which is likely tolead to neuron loss. Neuron function can be compromised by, for example,altered biochemistry, physiology, or anatomy of a neuron. Deteriorationof a neuron may include membrane, dendritic, or synaptic changes whichare detrimental to normal neuronal functioning. The cause of the neurondeterioration, impairment, and/or death may be unknown. Alternatively,it may be the result of age- and/or disease-related changes which occurin the nervous system of a subject.

When neuron loss is described herein as "age-related". it is intended toinclude neuron loss resulting from known and unknown bodily changes of asubject which are associated with aging. When neuron loss is describedherein as "disease-related", it is intended to include neuron lossresulting from known and unknown bodily changes of a subject which areassociated with disease. It should be understood, however, that theseterms are not mutually exclusive and that, in fact, many conditions thatresult in the loss of neurons are both age- and disease-related.

Exemplary age-related diseases associated with neuron loss and changesin neuronal morphology include, for example, Alzheimer's Disease, Pick'sDisease, Parkinson's Disease, Vascular Disease, Huntington's Disease,and Age-Associated Memory Impairment. In Alzheimer's Disease patients,neuron loss is most notable in the hippocampus, frontal, parietal, andanterior temporal cortices, amygdala, and the olfactory system. The mostprominently affected zones of the hippocampus include the CA1 region,the subiculum, and the entorhinal cortex. Memory loss is considered theearliest and most representative cognitive change because thehippocampus is well known to play a crucial role in memory. Pick'sDisease is characterized by severe neuronal degeneration in theneocortex of the frontal and anterior temporal lobes which is sometimesaccompanied by death of neurons in the striatum. Parkinson's Disease canbe identified by the loss of neurons in the substantia nigra and thelocus ceruleus. Huntington's Disease is characterized by degeneration ofthe intrastriatal and cortical cholinergic neurons and GABA-ergicneurons. Parkinson's and Huntington's Diseases are usually associatedwith movement disorders, but often show cognitive impairment (memoryloss) as well.

Age-Associated Memory Impairment (AAMI) is another age-associateddisorder that is characterized by memory loss in healthy, elderlyindividuals in the later decades of life. Crook, T. et al. (1986) Devel.Neuropsych. 2(4):261-276. Presently, the neural basis for AAMI has notbeen precisely defined. However, neuron death with aging has beenreported to occur in many species in brain regions implicated in memory,including cortex, hippocampus, amygdala, basal ganglia, cholinergicbasal forebrain, locus ceruleus, raphe nuclei, and cerebellum. Crook, T.et al. (1986) Devel. Neuropsych. 2(4):261-276.

Vitamin D3 compounds of formulas I and II can protect against neuronloss by genomic or non-genomic mechanisms. Nuclear vitamin D3 receptorsare well known to exist in the periphery but have also been found in thebrain, particularly in the hippocampus and neocortex. Non-genomicmechanisms may also prevent or retard neuron loss by regulatingintraneuronal and/or peripheral calcium and phosphate levels.Furthermore, vitamin D3 compounds of formulas I and II may protectagainst neuronal loss by acting indirectly, e.g., by modulating serumPTH levels. For example, a positive correlation has been demonstratedbetween serum PTH levels and cognitive decline in Alzheimer's Disease.

The present method can be performed on cells in culture, e.g. in vitroor ex vivo, or on cells present in an animal subject, e.g., in vivo.Vitamin D3 compounds of formulas I and II can be initially tested invitro using neurons from embryonic rodent pups (See e.g. U.S. Pat. No.5,179,109-fetal rat tissue culture), or other mammalian (See e.g. U.S.Pat. No. 5,089,517-fetal mouse tissue culture) or non-mammalian animalmodels. These culture systems have been used to characterize theprotection of peripheral, as well as, central nervous system neurons inanimal or tissue culture models of ischemia, stroke, trauma, nervecrush, Alheimer's Disease, Pick's Disease, and Parkinson's Disease,among others. Examples of in vitro systems to study the prevention ofdestruction of neocortical neurons include using in vitro cultures offetal mouse neurons and glial cells previously exposed to variousglutamate agonists, such as kainate, NMDA, andα-amino-3-hydroxy-5-methyl-4-isoxazolepronate (AMPA). U.S. Pat. No.5,089,517. See also U.S. Pat. No. 5,170,109 (treatment of ratcortical/hippocampal neuron cultures with glutamate prior to treatmentwith neuroprotective compound); U.S. Pat. Nos. 5,163,196 and 5,196,421(neuroprotective excitatory amino acid receptor antagonists inhibitglycine, kainate, AMPA receptor binding in rats).

Alternatively, the effects of vitamin D3 compounds of formulas I and IIcan be characterized in vivo using animals models. Neuron deteriorationin these model systems is often induced by experimental trauma orintervention (e.g. application of toxins, nerve crush, interruption ofoxygen supply). For example, in order to demonstrate that certainN-methyl-D-aspartate (NMDA), an excitatory amino acid neurotransmitterreceptor, antagonists were useful as anticonvulsants andneuroprotectants, the inventors in U.S. Pat. No. 4,957,909 employed amodel wherein Swiss-albino mice and rat hippocampal neurons weresubjected to overstimulation of excitatory amino acid receptorssubsequent to treatment with the NMDA antagonists. A similar study wasperformed wherein the utility of certain NMDA antagonists as agents thatprevent neurodegeneration was demonstrated by treating mice with NMDAsubsequent to treatment with the NMDA antagonists. U.S. Pat. No.5,168,103.

Smooth Muscle Cells

In yet another aspect, the present invention provides a method ofmodulating the activity of a vascular smooth muscle cell by contacting avitamin D3-responsive smooth muscle cell with a vitamin D3 compounds offormulas I or II to activate or, preferably, inhibit the activity of thecell. The language "activity of a smooth muscle cell" is intended toinclude any activity of a smooth muscle cell, such as proliferation,migration, adhesion and/or metabolism.

In certain embodiments, the vitamin D3 compounds of formulas I and IIcan be used to treat diseases and conditions associated with aberrantactivity of a vitamin D3-responsive smooth muscle cell. For example, thepresent invention can be used in the treatment of hyperproliferativevascular diseases, such as hypertension induced vascular remodeling,vascular restenosis and atherosclerosis. In other embodiments, thepresent invention can be used in treating disorders characterized byaberrant metabolism of a vitamin D3-responsive smooth muscle cell, e.g.,arterial hypertension.

The present method can be performed on cells in culture, e.g. in vitroor ex vivo, or on cells present in an animal subject, e.g., in vivo.Vitamin D3 compounds of formulas I and II can be initially tested invitro as described in Catellot et al. (1982), J. Biol. Chem. 257(19):11256.

This invention is further illustrated by the following examples which inno way should be construed as being further limiting. It is understoodby the ordinary skilled artisan that production of a vitamin D3 compoundof formula I or II in a cell is indicative that such compound isbiologically active in such cell, and thus that it can be used intreating conditions arising from aberrant activity of such cells. Forexample, production of vitamin D3 compounds of formulas I and II inkeratinocytes is indicative that such vitamin D3 compounds arebiologically active in those cells and can be used in treatingconditions such as psoriasis. The contents of all cited references(including literature references, issued patents, published patentapplications, and co-pending patent applications) cited throughout thisapplication are hereby expressly incorporated by reference.

EXAMPLES Example I

Isolation and Identification of a Cyclic Ether Metabolite of1α,25-Dihydroxy-Vitamin D₃ in Human Keratinocytes

As described herein, 1α25(OH)₂ -3-epi-D₃ is metabolized into a lesspolar metabolite than 1α25(OH)₂ -3-epi-D₃, peak M1, in humankeratinocytes (FIG. 2). FIG. 2 shows the HPLC profile and UV spectra ofthe metabolites produced in human keratinocytes incubated with 1α25(OH)₂-3-epi3D₃ (1 uM) for 24 H. On a straight phase HPLC system, thismetabolite (M1) is more polar than 25(OH)D₃ but less polar than1α25(OH)₂ D₃ and similar to that of 1α(OH)D₃. Mass spectrometricanalysis reveals a molecular ion of 414 m/z, which is 2 mass units lessthan the starting 1α25(OH)₂ D₃, shown in FIG. 3. FIG. 3 shows the massspectra of 1α(OH)₂ -3-epi-D₃ (M) (upper panel) and its cyclic ethermetabolite (M₁) (lower panel) isolated from human keratinocytesincubated with 1α25(OH)₂ -3-epi-D₃ (1 uM) for 24 h. The typicalfragments at m/z 134 and 152 m/z indicate an unmodified A-ring andcistriene structure. A double bond introduced at either C, D rings orthe side chain would be consistent with the molecular weight. However,this type of unsaturated metabolite still possesses a free 25-hydroxylgroup and is expected to have similar retention time as the startingcompound; this is contradicting to what was observed. Furthermore, theabsence of mass fragments at 59 m/z suggests the absence of a25-hydroxyl group. The absence of the familiar side chain cleavagefragments at 251, 269 and 287 m/z also suggests a modified 25-hydroxylgroup and a possible structural change at C-20 to retard the cleavage atcarbons 17 and 20. A cyclic structure as shown in FIGS. 3 and 4 issupported by these mass spectrometric and chromatographic evidences.This proposed structure is consistent with the loss of m/z 58 (acetone)to form m/z 356 and the subsequent fragments at 338, 320 and 314.

It is probable that the 3-epi modification of the A-ring allowsalternate side chain reactions to occur. FIG. 4 summarizes the proposedmetabolic pathway for the formulation of the cyclic ether metabolite of1α25(OH)₂ -3-epi-D₃. The formation of cyclic ether structure couldresult from a hydroxylation at either C-17 or C-20 and the subsequentreaction with the 25-hydroxyl group to form an ether linkage as shown inFIG. 4. This type of metabolic reactions are known to occur inhydroxylated fatty acids. Thus, it is probable that some of theunidentified metabolites can be C-17 or C-20-hydroxylated metabolites of1α25(OH)₂ -3-epi-D₃.

Example II

Isolation and Identification of a 3-Epi Metabolite of 1α Hydroxy-VitaminD₃ in Human Keratinocytes

FIG. 5A shows the metabolism of 1α(OH)-D₃ into its 3 epi form in theosteosarcoma cell line UMR-106. Peak A represents the 3-epi form of1α(OH)D₃. Peak B corresponds to the substrate, 1α(OH)D₃. The insertpanels show the UV spectra of the various metabolites as monitored byphotodiode array detector. FIG. 5B shows a schematic representation ofthe 3-epimerization of 1α(OH)D₃ into 1α(OH)-3-epi vitamin D₃. Similar to1α(OH)D₃, 1α(OH)-3-epi vitamin D₃ can be converted into the25-hydroxylated form in vivo.

1α(OH)D₃ compounds are currently used in the treatment of osteoporosis.Thus, 3-epi forms of these compounds may be used as substitutes for1α(OH)D₃ compounds in treating osteoporosis.

Example III

Confirmation of 3-epi Configuration of 1α(OH) 3-epi Vitamin D₃

To confirm the production of 1α(OH) 3-epi vitamin D₃ in bone cells. Themetabolites of 1α-3-epi-D₃ produced by the osteosarcoma cell line(UMR-106) were analyzed by mass spectroscopy. FIG. 6 shows the massspectra of 1α(OH)D₃ (upper panel) and its 3-epi metabolite (lowerpanel). A comparison of these two mass spectra revealed difference inpeaks observed only in the 3-epi metabolite, for example, fragmentshaving molecular weights of approximately m/z 57, 217, 312 and 529(lower panel). The mass spectrum of the 3-epi metabolite wasindependently confirmed to be 1α(OH) 3-epi vitamin D₃.

Example IV

Enhanced Stability In Vivo of 1α(OH) 3-Epi Vitamin D₃ Compared to itsIsomeric Counterpart

The stability of 1α(OH) 3-epi vitamin D₃ in vivo was characterized bymonitoring the changes in the concentration of 1α(OH) 3-epi vitamin D₃and its isomeric counterpart at various time intervals. In particular,FIG. 7 shows the HPLC profile and UV spectra of the metabolites producedin rat osteosarcoma cell lines (UMR-106) which were incubated with1α(OH)D₃ for 24, 48, or 84 hours. Peak M and S represent the relativeconcentrations of 1α(OH) 3-epi vitamin D₃ and its isomeric counterpart,respectively, at the time intervals tested. The persistent duration ofpeak M relative to peak S after 48 and 84 hour-incubations indicatesthat 3-epi metabolite of 1α(OH)D₃ are more stable in vivo than itsisomeric counterpart.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. An isolated 3-epi form of a 1α-hydroxy-vitamin D3compound having formula II as follows: ##STR8## wherein A₁ is a single,a double, or a triple bond; A₂, A₃ and A4 are each independentlyselected from the group consisting of a single or a double bond; R₂, R₃,R₄, R₇, R₈ and R₉ are independently selected from the group consistingof a hydrogen, a deuterium, a deuteroalkyl, a hydroxy, an alkyl, analkoxide, an O-acyl, a halogen, a haloalkyl, a hydroxyalkyl, an amine ora thiol group, and wherein the pairs of R₂ and R₃, and R₄ and R₇ takentogether are an oxygen atom; and R₅ and R₆ are independently selectedfrom the group consisting of a hydrogen, a deuterium, a halogen, analkyl, a hydroxyalkyl, a haloalkyl, and a deuteroalkyl.
 2. The compoundof claim 1, which is 1α(OH) vitamin D3, 1α,24 dihydroxy 3-epi vitaminD₃, 1α hydroxy 24-ethyl 3-epi vitamin D₃, 1α hydroxy 24-methyl 3-epivitamin D₃, or 1α, 24-dihydroxy 24-methyl 3-epi vitamin D₃.
 3. A methodof treating a disorder characterized by an aberrant activity of avitamin D₃ -responsive cell, comprising administering to a subject aneffective amount of a vitamin D₃ compound having formula II as follows:##STR9## wherein A₁ is a single, double, or a triple bond; A₂, A₃ and A₄are each independently selected from the group consisting of a single ora double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selected fromthe group consisting of hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl, such that the aberrant activity of the vitamin D₃-responsive cell is reduced.
 4. A method of treating a disordercharacterized by an aberrant activity of a hyperproliferative skin cell,comprising administering to a subject an effective amount of an isolated3-epi form of a 1α-hydroxy-vitamin D3 compound having formula II asfollows: ##STR10## wherein A₁ is a single, a double, or a triple bond;A₂, A₃ and A₄ are each independently selected from the group consistingof a single or a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ areindependently selected from the group consisting of a hydrogen, adeuterium, a deuteroalkyl, a hydroxy, an alkyl, an alkoxide, an O-acyl,a halogen, a haloalkyl, a hydroaxyalkyl, a amine or a thiol, and whereinthe pairs of R₂ and R₃, and R₄ and R₇ taken together are an oxygen atom;R₅ and R₆ are independently selected from the group consisting ofhydrogen, a deuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl,and a deuteroalkyl, such that the aberrant activity of thehyperproliferative skin cell is reduced.
 5. The method of claim 3,wherein the disorder comprises an aberrant activity of an endocrinecell.
 6. The method of claim 5, wherein the endocrine cell is aparathyroid cell and the aberrant activity is processing and/orsecretion of parathyroid hormone.
 7. A method of treating secondaryhyperparathyroidism, comprising administering to a subject an effectiveamount of an isolated 3-epi of a 1αhydroxy-vitamin D3 compound havingformula II as follows: ##STR11## wherein A₁ is a single, double, or atriple bond; A₂, A₃ and A₄ are each independently selected from thegroup consisting of a single or a double bond; R₂, R₃, R₄, R₇, R₈ and R₉are independently selected from the group consisting of hydrogen, adeuterium, a deuteroalkyl, a hydroxy, an alkyl, an alkoxide, an O-acyl,a halogen, a haloalkyl, a hydroxyalkyl, an amine or a thiol group, andwherein the pairs of R₂ and R₃, and R₄ and R₇ taken together are anoxygen atom; and R₅ and R₆ are independently selected from the groupconsisting pf a hydrogen, a deuterium, a halogen, an alkyl, ahydroxyalkyl, a haloalkyl, and a deuteroalkyl.
 8. A method of treating adisorder characterized by an aberrant activity of a bone cell,comprising administering to a subject an effective amount of an isolated3-epi form of a 1α-hydroxy-vitamin D3 compound having formula II asfollows: ##STR12## wherein A₁ is a single, double, or a triple bond; A₂,A₃ and A₄ are each independently selected from the group consisting of asingle or a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independentlyselected from the group consisting of hydrogen, a deuterium, adeuteroalkyl, a hydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, ahaloalkyl, a hydroxyalkyl, an amine or a thiol group, and wherein thepairs of R₂ and R₃, and R₄ and R₇ taken together are an oxygen atom; andR₅ and R₆ are independently selected from the group consisting pf ahydrogen, a deuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl,and a deuteroalkyl, such that the aberrant activity of the bone cell isreduced.
 9. The method of claim 8, wherein the disorder is selected fromthe group consisting of osteoporosis, osteodystrophy, senileosteoporosis, osteomalacia, rickets, osteitis fibrosa cystica, renalosteodystrophy, secondary hyperparathyrodism, cirrhosis, and chronicrenal disease.
 10. The method of claim 3, wherein the subject is amammal.
 11. The method of claim 10, wherein the mammal is a human.
 12. Amethod of ameliorating a deregulation of calcium and phosphatemetabolism, comprising administering to a subject a therapeuticallyeffective amount of a 3-epi vitamin D₃ compound of any of claims 1 or 2,so as to ameliorate the deregulation of the calcium and phosphatemetabolism.
 13. The method of claim 12, wherein the deregulation of thecalcium and phosphate metabolism leads to osteoporosis.
 14. Apharmaceutical composition comprising, a therapeutically effectiveamount of a vitamin D₃ compound of claim 1, and a pharmaceuticallyacceptable carrier.
 15. A method of treating osteoporosis, comprisingadministering to a subject an effective amount of an isolated 3-epi formof a 1α-hydroxy-vitamin D3 compound having formula II as follows:##STR13## wherein A₁ is a single, double, or a triple bond; A₂, A₃ andA₄ are each independently selected from the group consisting of a singleor a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selectedfrom the group consisting of hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.
 16. A method of treating osteodystrophy, comprisingadministering to a subject an effective amount of an isolated 3-epi formof a 1α-hydroxy-vitamin D3 compound having formula II as follows:##STR14## wherein A₁ is a single, double, or a triple bond; A₂, A₃ andA₄ are each independently selected from the group consisting of a singleor a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selectedfrom the group consisting of hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.
 17. A method of treating senile osteoporosis, comprisingadministering to a subject an effective amount of an isolated 3-epi formof a 1α-hydroxy-vitamin D3 compound having formula II as follows:##STR15## wherein A₁ is a single, double, or a triple bond; A₂, A₃ andA₄ are each independently selected from the group consisting of a singleor a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selectedfrom the group consisting of hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.
 18. A method of treating osteomalacia, comprisingadministering to a subject an effective amount of an isolated 3-epi formof a 1α-hydroxy-vitamin D3 compound having formula II as follows:##STR16## wherein A₁ is a single, double, or a triple bond; A₂, A₃ andA₄ are each independently selected from the group consisting of a singleor a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selectedfrom the group consisting of hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.
 19. A method of treating rickets, comprising administeringto a subject an effective amount of an isolated 3-epi form of a1α-hydroxy-vitamin D3 compound having formula II as follows: ##STR17##wherein A₁ is a single, double, or a triple bond; A₂, A₃ and A₄ are eachindependently selected from the group consisting of a single or a doublebond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selected from thegroup consisting of hydrogen, a deuterium, a deuteroalkyl, a hydroxy, analkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, a hydroxyalkyl,an amine or a thiol group, and wherein the pairs of R₂ and R₃, and R₄and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.
 20. A method of treating osteis fibrosa cystica,comprising administering to a subject an effective amount of an isolated3-epi form of a 1α-hydroxy-vitamin D3 compound having formula II asfollows: ##STR18## wherein A₁ is a single, double, or a triple bond; A₂,A₃ and A₄ are each independently selected from the group consisting of asingle or a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independentlyselected from the group consisting of hydrogen, a deuterium, adeuteroalkyl, a hydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, ahaloalkyl, a hydroxyalkyl, an amine or a thiol group, and wherein thepairs of R₂ and R₃, and R₄ and R₇ taken together are an oxygen atom; andR₅ and R₆ are independently selected from the group consisting pf ahydrogen, a deuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl,and a deuteroalkyl.
 21. A method of treating renal osteodystrophy,comprising administering to a subject an effective amount of an isolated3-epi form of a 1α-hydroxy-vitamin D3 compound having formula II asfollows: ##STR19## wherein A₁ is a single, double, or a triple bond; A₂,A₃ and A₄ are each independently selected from the group consisting of asingle or a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independentlyselected from the group consisting of hydrogen, a deuterium, adeuteroalkyl, a hydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, ahaloalkyl, a hydroxyalkyl, an amine or a thiol group, and wherein thepairs of R₂ and R₃, and R₄ and R₇ taken together are an oxygen atom; andR₅ and R₆ are independently selected from the group consisting pf ahydrogen, a deuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl,and a deuteroalkyl.
 22. A method of treating cirrhosis, comprisingadministering to a subject an effective amount of an isolated 3-epi formof a 1α-hydroxy-vitamin D3 compound having formula II as follows:##STR20## wherein A₁ is a single, double, or a triple bond; A₂, A₃ andA₄ are each independently selected from the group consisting of a singleor a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selectedfrom the group consisting of hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.
 23. A method of treating chronic renal disease, comprisingadministering to a subject an effective amount of an isolated 3-epi formof a 1α-hydroxy-vitamin D3 compound having formula II as follows:##STR21## wherein A₁ is a single, double, or a triple bond; A₂, A₃ andA₄ are each independently selected from the group consisting of a singleor a double bond; R₂, R₃, R₄, R₇, R₈ and R₉ are independently selectedfrom the group consisting of hydrogen, a deuterium, a deuteroalkyl, ahydroxy, an alkyl, an alkoxide, an O-acyl, a halogen, a haloalkyl, ahydroxyalkyl, an amine or a thiol group, and wherein the pairs of R₂ andR₃, and R₄ and R₇ taken together are an oxygen atom; and R₅ and R₆ areindependently selected from the group consisting pf a hydrogen, adeuterium, a halogen, an alkyl, a hydroxyalkyl, a haloalkyl, and adeuteroalkyl.